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Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Introduction

It is more than 10 years after the completion of the draft sequence of the human genome was

published in Nature and more than 140 years after the discovery of nucleic acids. During this time
molecular biology was borne and became the most important branch of biology and probably
the most important science today. In 1961, William Astbury described molecular biology as ”not so
much a technique as an approach, an approach from the viewpoint of the so-called basic sciences
with the leading idea of searching below the large-scale manifestations of classical biology for the
corresponding molecular plan. It is concerned particularly with the forms of biological molecules and
[...] is predominantly three-dimensional and structural – which does not mean, however, that it is
merely a refinement of morphology. It must at the same time inquire into genesis and function.”

Modern molecular biology describes biological systems on the molecular level, describes

molecules,  mainly  proteins  and  nucleic  acids.  Research  conducted  on  nucleic  acids  formed
fundaments for modern molecular biology and modern science. The development of this knowledge
was only possible due to the development of techniques allowing the analysis of molecules in cells.
A variety of methods was discovered allowing the identification of proteins and nucleic acids, analysis
of  their  sequence  and  structure,  as  well,  and  probably  most  importantly,  interactions  between
proteins and nucleic acids. The discovery of methods for DNA sequencing, amplification and analysis
were honored by the Nobel committee, showing the importance of those discoveries. This amazing
development  of  molecular  biology  techniques  allows  the  accumulation  of  new  knowledge  on
the structure and functioning of biological systems. The results of molecular biology experiments are
important nowadays for substantially all biology branches such as microbiology, structural biology,
developmental biology, neurobiology, physiology, ecology and many others. Molecular biology forms
the  bases  and  is  a  part  of  medical  sciences  and  pharmacology.  Drug  discovery,  development  of
modern  therapies  would  not  be  possible  without  molecular  biology,  it’s  techniques  and  data
accumulated  from  thousands  of  experiments  conducted  day  after  day  in  laboratories  all  over
the world. That data and the discovery and development of new techniques are bringing progress to
our  understanding  of  living  organisms  and  allow  the  implementation  of  this  knowledge  for  our
benefit.

Here we present a brief description of selected basic techniques used in molecular biology

for the  analysis  of  nucleic  acids.  The  described  techniques  are  important  for  understanding  basic
concepts of molecular biology and understanding complex technology and experimental approaches.
Studying  molecular  biology  methods  brings  the  understanding  of  their  bases  and  appreciation  of
the intellectual input standing behind their discovery and development. Also, it brings the ability to
understand  results  described  in  science  publications.  The  understanding  of  the  fundaments  of
particular techniques is critical for their proper application. The presented selection will be boarded
and  permanently  modified  to  create  the  most  suitable  tool  for  students  during  molecular  biology
studies with particular emphasis on nucleic acids. The selection shows principles for basic techniques
analyzing nucleic acids, difficulties in the application of these techniques and possible applications of
particular  methods.  At  the  end  of  each  chapter  the  reader  can  find  further  literature  for  reading
and questions which should help with deeper understanding of the presented matter.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Important dates and facts in the history of molecular biology

1869  Fritz Miescher discovers that the pus cells contain an substance to which he gave the name 'nuclein'
1871  Discovery of nucleic acids
1910  Discovery that genes lie on chromosomes
1913  Chromosomes are linear arrays of genes
1919  Phoebus Aaron Levene proposes the 'tetranucleotide' structure of DNA
1927  Mutations are physical changes in genes
1928  Frederick Griffith described in bacteria the phenomenon 'transformation'
1931  Discovery of crossing over recombination
1938  Rudolf Signer, Torbjorn Caspersson and Einer Hammarsten estimated molecular weights for DNA between

500 000 and 1 000 000 daltons

1944 Oswald Avery, Colin MacLeod and Maclyn McCarty described that Griffith's transforming is DNA based.

They proposed that DNA may be the genetic material

1949 Erwin Chargaff proposed that DNA base composition varies from one species to another and proposed the

so called Chargaff’s rules

1949 Roger and Colette Vendrely and André Boivin find that half as much DNA in the nuclei of sex cells is

present in the body cells

1951  Rosalind Franklin distinguishes two forms of DNA
1952  Discovery that viral DNA but not proteins enters the bacterial cell (Al Hershey and Martha Chase)
1952  X-ray diffraction pattern of the B form of DNA (Rosalind Franklin and Raymond Gosling)
1953  James Watson and Francis Crick, Rosalind Franklin and Raymond Gosling, Maurice Wilkins, W. E. Seeds,

Alec Stokes and Herbert Wilson, and Bertil Jacobson publish on the structure of DNA

1954 George Gamow proposed that DNA code for the synthesis of proteins
1955 Seymour Benzer analyses the structure of DNA of a bacterial virus close to the distances of the individual

bases

1957  The ‘sequence hypothesis' and 'the central dogma' (Francis Crick)
1958  Matthew Meselson and Franklin Stahl demonstrate that DNA replication is semi-conservative
1959  Arthur Kornberg isolates the enzyme DNA polymerase
1961  Description  that  a  sequence  of  nucleotides  encodes  a  particular  amino  acid.  The  genetic  code  was

depicted (Marshall Nirenberg and Johann Heinrich Matthaei)

1961  mRNA discovery, “Operon Model” by Jacob and Monod
1962  The Nobel prize in medicine is awarded to James Watson, Francis Crick and Maurice Wilkins
1963  “Replicon Model” by Jacob and Brenner
1967  The antisense RNA
1968  The discovery of restriction enzymes (Arber, Smith, Natans)
1972  The introduction of recombinant DNA technology (Boyer, Cohen)
1977  The antisense technology (Zamecnik)
1977  DNA sequence analysis methods (Sanger, Maxam, Gilbert)
1979  Z-DNA form is discovered by Rich
1982  National Institute of Health established GeneBank
1983  Polymerase Chain Reaction developed by Mullis
1990  The beginning of the Human Genome Project
1995  H. influenza genome
1996  Sacharomyces cerevisiae genome
1998  C. elegance genome
2001  Human genome sequence published
2007  The beginning of high speed modern DNA sequencing technology

ITERATURE

Astbury W.T, 1961, Molecular Biology or Ultrastructural Biology? Nature 190: 1124
The Human Genome at Ten, 2010 Nature 470
The double helix – 50 years. 2003, Nature 421

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 1. Polymerase chain reaction (PCR)

The polymerase chain reaction is one of the most popular techniques in molecular biology

(e.g. for amplification particular DNA fragment designed for cloning), diagnostic (e.g. for analysis of
point mutations), forensic sciences (e.g. for affiliating fatherhood) etc. It was developed in 1983 by
Kary Mullis, who in 1993 was awarded the Nobel Prize in chemistry. The polymerase chain reaction
enables the amplification of the initial amount of nucleic acid, which increases exponentially and can
be calculated from the formula:

N = N

× 2

where N is the amount of the product of the reaction

is the amount of the template

n is the number of cycles

Unfortunately this formula does not take into account the reaction efficiency. If the reaction
efficiency is E, it can be added to the formula:

N = N

× (1 + E)

At the beginning of the reaction in the initial cycles, the reaction efficiency is usually 0.8-0.97 and it
decreases parallel with the accumulation of the reaction product, with the decrease of nucleotides
and primers and also with the decrease of enzyme activity.
In order for the PCR reaction to take place, the reaction mixture has to contain:

− DNA template (theoretically just one DNA molecule is sufficient to obtain amplification product.

Usually the amount of 1-10ng bacterial DNA, 0.1-1ng plasmid DNA or up to 500ng human genomic
DNA is used as a template)

− polymerase (the enzymes used in PCR are isolated from thermostabile bacteria and their

optimum temperature is around 70°C. Depending on the amplification product destination
a polymerase with different processivity and fidelity is chosen. The most common one is Taq
polymerase obtained from Thermus aquaticus. Since it is characterized by high processivity but
lower fidelity it is used to obtain high amount of the PCR product. If high fidelity is needed then
the Pfu polymerase from Pyrococcus furiosus or Pwo from Pyrococcus woesei are usually used.
For a standard 50 µl reaction 1U of enzyme is used)

− primers (they are short DNA fragments, usually around 25bp, that contain 40-60% of GC residues.

They are complementary to the particular regions within the DNA template, that flank the region
which is going to be amplified. Primer melting temperature (Tm) should be the same for both of
them and is usually around 50-60°C. It is important that they do not form secondary structures
and are not complementary to each other. The concentration of both of them should be around
0.2-1 µM)

− Mg

(this ion is a polymerase cofactor. Its concentration should be around 2mM but very often

has to be estimated individually for a particular PCR experiment. The excess of Mg

can inhibit

the PCR reaction by interaction with nucleotides, what decreases the amount of available
nucleotides in the reaction mixture)

− dNTPs (they are dATP, dTTP, dGTP, dCTP. The concentration of each of them should be up to

200 µM and should not be exceeded, since the excess of dNTPs can result in a unspecific reaction

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

product and can chelate the Mg

ions. On the other hand, a too low concentration of dNTPs

results in quick arrest of the reaction via the depletion of reaction components)

− buffer (the standard buffer for the PCR reaction contains 10-50mM Tris pH 8-9 and sodium

or potassium salts)

Fig. 1. Scheme of the first and second cycle during the polymerase chain reaction. The template used in the

reaction is presented in black, primers and synthesized complement strand are presented in red.

Three main steps can be distinguished in the PCR reaction (Fig. 1):
1. Denaturation - it is the first step during which by heating the reaction mixture to 95°C for 30-120

sec. the DNA template melts)

2. Annealing - it is a step when the primers hybridize to the complement strand of DNA template.

It is carried out for 30-60 sec. in a temperature that depends on the melting temperature (Tm)
of primers. The simplest formula to calculate the Tm takes into account the number of AT and GC
residues within the primer

Tm = 2°C × (A+T) + 4°C × (G+C)

The annealing temperature is usually 5°C lower than the Tm, however it should be optimized for a
particular pair of primers.

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

denaturaion

annealing

elongation

the first cycle

denaturation

denaturation

5’

3’

5’

3’

5’

3’

5’

3’

annealing

5’

3’

5’

3’

5’

3’

5’

3’

elongation

5’

3’

5’

3’

5’

3’

5’

3’

next cycles

second cycle

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

denaturaion

annealing

elongation

the first cycle

denaturation

denaturation

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

annealing

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

elongation

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

5’

3’

next cycles

second cycle

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

3. Elongation - in this step the primers are elongated and nucleotides complementary to

the template are added. The elongation temperature depends on the optimum temperature of
the enzyme and the time of this process depends on the processivity of polymerase. For instance
Taq polymerase can add 1000 nucleotides in 1 min and for Pfu polymerase it takes 2 min.
The elongation time should be applied properly since a too long elongation can result in
unspecific products and too short can lead to no product formation).

These three steps are repeated 20-30 times, one after the other as a rule. Usually the first cycle is
preceded  by  initial  denaturation  in  95°C  for  up  to  2  min  and  the  last  one  is  followed  by  terminal
elongation in 72°C for 5 min.
The PCR products are then further analyzed by electrophoresis.

BRIDGED

PCR

PROGRAM

Temperature

Time

1. 95°C

30 sec

2. 95°C

30 sec

3. Tm-5°C

30 sec

4. 72°C

time (sec) = (number of base pairs/ 1000 for Taq) × 60

Steps from 2 to 4 are repeated 20-30 times

5. 72°C

30 sec

ROUBLESHOOTING

1. If there is no PCR product:

−  the temperature of primer annealing should be decreased
−  the elongation time should be increased
−  the Mg

concentration should be optimized

−  the dNTPs concentration should be increased
−  the DNA template concentration should be checked
−  the formation of secondary structures by primers should be checked
−  the primers concentration should be checked

2. If shorter unspecific products are present

−  the elongation time should be increased
−  the temperature of primer annealing should be increased
−  the contamination of the reaction mixture components should be checked

3. If longer unspecific products are present

−  the elongation time should be decreased
−  the concentration of dNTPs and primers should be decreased
−  the temperature of primer annealing should be increased
−  the contamination of the reaction mixture components should be checked

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Variety of the method

PCR-RFLP (PCR- Restriction Fragment Length Polymorphism)- is a technique, in which the DNA from
different species e.g. the wild type and the mutant, is amplified and the PCR products are digested
with restriction enzymes. The electrophoretic analysis of obtained restriction fragments results with
restriction  profile  of  the  organism  (DNA  fingerprint).  The  profiles  can  be  compared  between
the organisms  and  show  the  differences  in  restriction  sites  and  therefore  the  differences  in
the sequences.

PCR-SSCP (PCR-Single Strand Conformation Polymorphism) – the first step in PCR-SSCP is the classical
PCR reaction, which is followed by denaturation of the obtained amplification products. The received
ssDNA is then separated in a gel during electrophoresis. Because various ssDNA can form different
secondary structures, ssDNA molecules with the same length can migrate differently in the gel. The
distinctions in the migration can be observed even for the molecules that differ in just one
nucleotide. Therefore the PCR-SSCP can be utilized for mutational analysis.

RAPD-PCR (

RT-PCR (Reverse Transcription - PCR) – is the PCR reaction during which the RNA is transcribed into
cDNA  by  reverse  transcriptase.  The  cDNA  can  be  used  then  as  a  template  in  classical  PCR.  Both
reverse  transcription  and  classical  PCR  can  be  performed  either  in  one  tube  or  separately.
The transcription  of  RNA  into  cDNA  occurs  at  40-50°C  depending  on  the  reverse  transcriptase,
further DNA amplification is performed according to the standard PCR protocol. The method can be
useful during gene library preparation and other techniques where cDNA is utilized.

Random Amplification of Polymorphic DNA – PCR) – is a PCR reaction in which the

amplifying region is essentially unknown. It utilizes just one short (10-20 nucleotides) random primer
with high content of GC residues. In first two cycles of RAPD-PCR, the primer annealing is performed
in  low  temperature  (35-45°C),  allowing  the  random  hybridization  of  the  primer,  even  to  not  fully
complement sequences of the template. In the following cycles, the annealing occurs in temperature
characteristic  for  the  primer.  Since  DNA  of  different  organisms  is  not  identical,  the  band  pattern
formed  by  RAPD-PCR  products  in  agarose  electrophoresis  can  be  utilized  to  distinguish  them  and
evaluate the relationship between the organisms.

Real-time PCR – is the PCR reaction during which the increasing amount of the reaction product is
monitored  in  real  time.  There  are  common  methods  of  detection  of  reaction  progression:  using
fluorescent dye like Sybr Green or TaqMan probes. The first one utilizes the dye, which intercalates
the dsDNA. Since the PCR product is intercalated by the fluorescent dye, the more product the more
fluorescence  detected.  In  the  second  method,  the  TaqMan  probes  (  short  DNA  fragment  with
a fluorescent tag at 5’ and a quencher at 3’ end respectively) anneal to the amplifying region and are
degraded  by  polymerase  during  elongation  of  the  primer.  The  degradation  of  the  probe  causes
the separation  of  the  fluorescent  tag  from  the  quencher,  what  results  with  fluorescence  of
the fluorophore. Therefore the more amplification reactions the more fluorescence detected. One of
the most important parameters of real-time PCR is threshold cycle (C

), indicating the cycle at which

the  instrument  can  distinguish  the  amplification  generated  fluorescence  from  the  background.
It depends  on  the  initial  amount  of  DNA  template,  since  the  higher  number  of  DNA  template
molecules  the  lower  C

. The estimation of investigated DNA copy number requires the standard

curve tracing, which is the C

plotted against template copy number (Fig. 2B). To trace the standard

curve the PCR reaction with a different known copy number of DNA template should be performed.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

As a result the curves showing the dependence between the fluorescence and cycle numbers are
obtained and for each curve the C

value can be read out (Fig. 2A). Then the C

values are plotted

against template copy number resulting with standard curve (Fig. 2B).

Fig 2. Real-time PCR curves. (A) The graph shows the curves of detected fluorescence dependence from

number of cycles. The addition of higher amount of a template results with lower cycle number in which

the thermocycler can detect the fluorescence. The green curve represents the reaction with the lowest

template amount and the black curve with the highest one. A thick black line shows the threshold, which is

a fluorescence above which the instrument can distinguish the amplification generated fluorescence from

the background. (B) The standard curve determined during real-time PCR reactions with increasing known

amounts of a template. The curve shows the dependence of threshold cycle C

from a template copy numbers.

When the standard curve is traced, next the final experiment of template copy number
determination in the investigated samples can be performed. After the real-time PCR reaction with
investigated samples as a template, the threshold cycle can be obtained. Then the C

value serves to

read off the template copy number from the standard curve.

Emulsion PCR – is a classical PCR reaction but performed in a drop of water-in-oil emulsion, what
facilitates  the  amplification  of  genomic  libraries  and  cDNA  libraries.  It  requires  the  preparation  of
an oil-surfactant  mixture  (e.g.  mixture  of  Span80,  Tween  80,  Triton X-100  and  mineral  oil)  and
aqueous phase, which is the classical PCR reaction mixture. Then the aqueous phase is added to the
oil phase in a dropwise manner and the mixture should be stirred for few minutes. After that the PCR
reaction  can  be  performed  and  when  it  is  finished  the  amplification  product  should  be  extracted
from  the  emulsion.  This  kind  of  PCR  is  used  for  instance  during  next-generation  of  sequencing
methods such as Sequencing by Oligonucleotide Ligation and Detection (SOLiD).

cycle number

uor

enc

threshold

1 C

2 C

3 C

cycle number

uor

enc

threshold

1 C

2 C

3 C

template copy number

thr

hol

num

ber

)

template copy number

thr

hol

num

ber

)

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

ITERATURE

Bartlett J. M. S. & Stirling D, 2003, PCR protocols, Methods Mol. Biol. 226, Humana Press.
Dieffenbach C.W. & Dveksler G.S, 2003, PCR primer: a laboratory manual. Second edition, Cold Spring Harbor

Laboratory Press.

Williams R, Peisajovich S.G, Miller O.J, Magdassi S, Tawfik D.S, Griffiths A.D, 2006, Amplification of complex

gene libraries by emulsion PCR, Nature Methods 3(7): 545-550.

McPherson M. J. and Møller S. G, 2006, PCR. Second edition, Taylor & Francis Group.

UESTIONS

1. What kind of PCR would you perform during:

a. plasmid analysis
b. copy number estimation
c. mutational analysis
d. library preparation

2. What is the difference between the first and the next cycles of PCR reaction?
3. Figure shows the result of real-time PCR (A). Based on the standard curve (B) determine the template copy

number within the analyzed sample.

cycles

uor

enc

threshold

cycles

uor

enc

threshold

template copy number

thr

hol

num

ber

template copy number

thr

hol

num

ber

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 2. DNA sequencing

DNA sequencing started almost forty years ago. In this time the methods developed in 1970s

by Sanger or Maxam and Gilbert were improved many times and they are still perfected to read as
many DNA sequences as fast as possible. The automation of sequencing as well as the application of
new solutions (Polony sequencing, pyrosyquencing, Illumina sequencing, SOLiD sequencing)
remarkably reduce labor time and make this process easier, more accurate and reliable.

Maxam-Gilbert sequencing method

Two scientist from Harvard University, Allan Maxam and Walter Gilbert in 1977 developed the

sequencing method called chemical sequencing. It is based on chemical degradation of the 5’-end
radiolabeled  DNA  fragment  of  interest.  The  DNA  can  be  either  single  or  double  stranded  and
the cleavage  reactions  are  performed  in  four  tubes.  To  each  of  them  a  diverse  cleavage  agent,
modifying different nucleotide and therefore diverse in cutting position, is added. The action of the
chemical is based on three steps: modification of the nitrogenous base by the chemical, removal of
the base from the sugar and breaking of the DNA at the sugar point (often supported by adding hot
piperidine to the sample). After the cleavage reactions, the obtained DNA fragments are separated in
a polyacrylamide gel, resolution of which is the main limitation of this sequencing method. Because
the  DNA  utilized  in  the  reactions  is  labeled,  the  visualization  of the  bands  is  easy  with the  use of
autoradiography and the sequence can be read based on the analysis of all the bands (Fig. 3).
Cleavage agents:

− dimethyl sulfate (DMS) - cleaves preferentially guanine (G) through its methylation, causing

destabilization of a glycosidic bond; in some conditions it can also cut adenine (A+G)

−  acid (e.g. formic acid) – cleaves both adenine and guanine (A+G)
−  dimethyl sulfate with acid or alkali - cleaves adenine (A)
−  hydrasine – cleaves the cytosine and thymine (C+T)
−  hydrasine with 2M NaCl - cleaves the cytosine

Fig. 3. DNA sequencing gel after Maxam-Gilbert sequencing

method. The products of four degradation reactions are

separated in four lanes. In every reaction a different cleavage

agent is used that preferentially degrades glycosidic bond

between sugar and nitrogenous base: guanine (G), guanine or

adenine (G+A), cytosine or thymine (C+T) and cytosine (C).

Sanger sequencing method

The method was developed in 1977 by Frederick Sanger’s group and was first used for

sequencing of the ΦX174 bacteriophage genome. The Sanger method, also called
the chain-termination method is based on termination of the DNA synthesis reaction after
the incorporation of a dideoxynucleotide triphosphate (ddNTP). The DNA synthesis is performed in

G+A

C+T

T
C
G
C

A
C
G

G+A

C+T

T
C
G
C

A
C
G

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

four  separate  tubes,  containing,  besides  the  DNA  template,  a  primer  and  a  DNA  polymerase,  one
ddNTP  triphosphate  (ddATP,  ddTTP,  ddGTP  or  ddCTP)  and  three  remaining  deoxynucleotide
triphosphates  (dNTP).  Since  the  modified  nucleotides  do  not  contain  a  3’-OH  group,
the phosphodiester bond cannot be formed between the ddNTP and the next dNTP. In each tube the
reaction  is  stopped  in  a  position  of  a  nucleotide  with  different  nitrogenous  base  A,  T,  G  or  C,
depending on the ddNTP added. Because of the small amount of the ddNTP in reactions the chain
termination occurs rarely and stochastically ( statistically once per each template DNA). The products
of  the  synthesis  reaction  are  separated  in  a  poliacrylamide  gel  and  a  visualization  of  the  DNA
fragments  is  achieved  due  to  the  use  of  radio-  or  fluorescent-  labeled  ddNTPs  (Fig.  4).  A  labeled
primer or dNTPs can also be used in the reaction.

Fig. 4. DNA sequencing gel after Sanger sequencing method.

The products of four DNA synthesis reactions separated in

four lanes. In every reaction different a ddNTP is added to

the reaction mixture: ddGTP (G), ddATP (A), ddTTP (T) or

ddCTP (C). When the ddNTP is incorporated into the DNA

chain the synthesis of DNA stops.

The Sanger method was the base for automated sequencing, where the synthesis reaction

products are separated by capillary electrophoresis and fluorescence is read by a detector. Currently,
automated sequencing is still being developed to obtain lower costs and higher throughput.
The challenge was also the miniaturization of the system and microchip capillary electrophoresis has
been successfully applied, what reduced the time and the reagents' volumes used and made possible
signal detection from a single molecule template.

Pyrosequencing method

This method is based on the synthesis sequencing method developed in late 1990s. It utilizes

detection of a pyrophosphate (PPi) released during a DNA polymerization reaction. The detection of
this process requires the cascade of enzymatic reactions, presented as equations:

(DNA)

+ dNTP (DNA)

n+1

+ PPi

PPi + APS ATP + SO

ATP + luciferin + O

AMP + PPi + oxyluciferin + hv

where APS stands for adenosine 5’-phosphosulphate and hv represents an emitted photon.

In the first step, one of four dNTPs is added to the reaction and if it is incorporated to

the chain, the enzymatic cascade is triggered and a photon of light is emitted, what is detected by
a sensor and marked as a peak on the pyrogram (diagram showing detected light depending on

T
C
G
C

A
C
G

T
C
G
C

A
C
G

luciferase

ATP

sulphurylase

DNA

polymerase

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

the added  dNTP)  (Fig.  5).  If  the  added  dNTP  is  not  complementary  to  the  nucleotide  within
the template  the  light  is  not  emitted.  In  the  next  step  the  second  dNTP  is  added  and  so  on.
The procedure is repeated as long as the whole sequence is read.

The pyrosequencing reactions can be performed as a solid-phase reaction or liquid-phase

sequencing. The first one requires washing of the non-incorporated nucleotides therefore the DNA
template  is  immobilized  on  a  solid  support  such  as  magnetic  beads  to  avoid  signal  decrease.
The second  one  omits  the  step  of  washing  of  the  non-incorporated  nucleotides  by  addition  of
a fourth enzyme e.g. apyrase, that degrades the free nucleotides.

Fig. 5. Pyrogram. The diagram shows dependence

between detected light and added dNTP. The sequence

reads from the pyrogram what is written in red below

the diagram.

Pyrosequencing became a very popular method of reading the DNA sequence since

it eliminates  the  necessity  of  primer  or  dNTPs  labeling  and  performing  electrophoresis.  It  also
reduces  time  and  enables  real-time  detection.  Unfortunately,  it  also  has  some  disadvantages  like
the necessity  of  the  washing  step  in  the  solid-phase  sequencing  or  the  decrease  of  the  activity of
apyrase  and  its  interaction  with DNA  in  liquid-phase method.  It can  also  bring  some  difficulties  in
reading the proper number of the same nucleotide in homopolymeric regions where a nucleotide is
repeated more than five times due to nonlinear light response.

Sequencing by Oligonucleotide Ligation and Detection (SOLiD) sequencing

method

Sequencing by Oligonucleotide Ligation and Detection is a technique of sequencing developed

in the last few years by the Life Technology company. It has a great throughput and enables reading
of 3-4Gb  in  approximately  5  days.  SOLiD  method  consist  of  four  processes  following  one  after
another. It starts from preparation of a library of the DNA of interest and specific adaptors (a short
DNA  fragments).  The  adaptors  are  ligated  to  the  DNA  fragments  and  hybridize  to  the  adaptor
complementary  sequences  immobilized  on  beads.  This  facilitates  the  deposition  of  a  lot  of  DNA
fragments  to  a  bead.  Then  an  emulsion  PCR  (see  Chapter 1)  is  performed  and  the  clonal bead

itte

lig

nucleotide added

nucleotide sequence

itte

lig

nucleotide added

nucleotide sequence

nucleotide added

nucleotide sequence

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

populations  are  formed.  Next,  the  beads  are  depositioned  on  a  glass  slide.  After  that,  the  actual
sequencing  reaction  can  be  performed  (Fig.  6).  During  the  sequencing  reaction  the  elongation  of
primer,  that  is  hybridized  to  the  adaptor  DNA  occurs.  The  fluorescently  labeled,  at  the  3’end,  di-
based  probes  are  added  (the  probes  are  8  bp  oligonucleotides)  and  just  the  ones  that  contain
complementary  sequences  hybridize  to  the  sequencing  DNA  (Fig.  6A).  Next  the  non-hybridized
probes are washed and the probe that hybridizes to the template is ligated to the primer (Fig. 6B).
Since the probes differ in the first two nucleotides and are labeled with different fluorescent dyes,
after the cycle the probe which hybridized to the template and which ligated to the primer can be
identified. Therefore from the first cycle the first two nucleotides of the sequence are read. After
the ligation reaction and reading the fluorescence, the two nucleotides at the 3’ end of the probe are
cleaved (Fig. 6C) and then the next cycle can be performed. In the next cycle consecutive di-bases are
added and the seventh and eighth nucleotides of the sequence are read.

Fig. 6. The first cycle of sequencing

reaction during SOLiD sequencing

method. After the hybridization of

the primer to the adaptor sequence,

the different fluorescently labeled

di-based probes are added (A).

When the complement de-base

hybridizes to the template, the

ligase support its ligation to the

primer (B). The fluorescence is read

and then the last two nucleotides of

the probe are cleaved and removed

(C). After that the next cycle can be

performed.

In the following cycles the ligation of the probes enable the reading of every fifth and sixth

nucleotide. To read the whole sequence, primers that differ in length are used. If the length of
the first used primer is n, the length of the next one could be n-1, then n-2 and so on. This method
enables to over read the same sequence what improves the reliability of the method and because
high amounts of DNA fragments can be attached to one bead via an adaptor sequence, it enables
the preparation of a lot of sequencing reactions on a small surface.

bead

ligase

adapter

primer

G T

A C

G G

adapter

primer

adapter

primer

ligation

cleavage

bead

ligase

adapter

primer

G T

A C

G G

adapter

primer

adapter

primer

ligation

cleavage

bead

ligase

adapter

primer

G T

A C

G G

adapter

primer

adapter

primer

ligation

cleavage

bead

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

ITERATURE

Franca L.T.C, Carrilho E, Kist T.B.L, 2002, A rewiev of DNA sequencing Techniques, Quarterly Reviews of

Biophysics 35(2): 169-200.

Graham C.A, Hill
Hindley J, Staden R, 1983, Laboratory Techniques in Biochemistry and Molecular Biology. DNA sequencing Vol.

10, Elsevier Biomedical.

A.J.M, 2001, DNA sequencing protocols, Humana Press Inc.

Janisz M, 2008,

Mardis. E. R, 2007, The impact of the next-generation sequencing technology on genetics. Trends in Genetics

24(3): 133-141.

Next-generation genome sequencing: towards personalized medicine, Wiley-Vch Verlag GmbH

& Co.

Maxam A. M, Gilbert M, 1977, A new method for sequencing DNA, Proc Natl Acad Sci USA 74(2): 560-564.
Michelson K.R, 2007, New high throughput technologies for DNA sequencing and genomics, Elsevier.

www.appiledbiosystems.com

UESTIONS

1. The figure shows the result of a sequencing reaction. What sequencing method was used? What was the

sequence of the analysed DNA fragment?

2. What kind of sequencing method would you choose to read the sequence of the bacterial genome in just a

few days? Explain your choice.

3. Which sequencing methods are based on fluorescence detection?

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 3. Gel Mobility Shift Assay

One of the simplest methods utilized to investigate interactions between nucleic acids and

proteins is the gel mobility shift assay (GMSA). The nomenclature of this technique is different and
names such as electrophoretic mobility shift assay (EMSA), gel shift assay or gel retardation assay are
used. Originally, GMSA was exploited to analyze rRNA-protein interactions and the action of E. coli
lactose operon regulatory system (Dahlberg et al, 1969; Fried & Crothers, 1981). The basis of
the method is a difference in the migration of naked DNA and a protein-DNA complex in an agarose
or poliacrylamide gel (Fig. 7). When a protein is bound to a DNA fragment, we observe a retardation
of a DNA band migration in a gel, compared with the migration of a naked nucleic acid.

Fig. 7. Scheme of GMSA in titration of

a labeled DNA fragment with increasing

amounts of a protein. All samples contain

equal amounts of DNA (load DNA) and

increasing amounts of the protein. The more

protein was added to the sample the more

electrophoretic bands were observed.

Schematic presentation of the electropho-

retic species is shown on the right.

Since every nucleoprotein complex is different, before carrying out the GMSA, the target (nucleic
acid), the labeling method, the binding and electrophoresis conditions should be carefully selected.

Nucleic acids

Choice of the appropriate fragment of the nucleic acid (DNA or RNA), its length should be

taken  into  consideration.  Usually,  short  fragments  of  nucleic  acids  are  used  in  the  GMSA
experiments. The shorter the fragment the higher electrophoretic resolution of the complexes from
free  nucleic  acid  and  the smaller the  number of  non-specific  protein  binding  sites.  However, with
shorter nucleic acid fragments the “end-effect” can occur, which results in aberrant protein binding.
Lengthening of the fragments can help in avoiding this limitation but can also cause such problems as
non-specific binding or smaller resolution of the bands.

When the length of the target DNA fragment is chosen then the labeling method should be

selected. To label and then to visualize the nucleic acids, radioisotopes, fluorophores or haptens can
be used. The most popular method is to label the 5’ or 3’ end with

P-phosphate, since it offers very

high  sensitivity  (the  subpicomole  range)  and  does  not  change  the  structure  of  the  nucleic  acid.
To label  DNA  with  radioisotope  incorporation  of  a  [γ-³²P]dNTP  during  a  3'-end  filling  reaction  by
Klenow fragment of DNA polymerase I or 5'-end labeling using the [γ-³²P]ATP and T4 polynucleotide
kinase can be performed.

An alternative to radioisotopes are fluorescent dyes and tags. The examples of dyes enabling

detection of dsDNA without any changes in the migration pattern are the phenanthridinium dimer,
ethidium homodimer (EthD) or the asymmetric cyanine compounds like the thiazole orange
homodimer (TOTO) and the oxazole yellow homodimer (YOYO). Also, dyes like Sybr Gold, Sybr Green
I and II could be advantageous in the GMSA, but during their use the DNA should be stained post

Protein

Probe

(load DNA)

Protein

Probe

(load DNA)

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

electrophoresis  due  to  the  possible  alterations  in  the  nucleic  acid  migration  in  a  gel.  When  using
fluorophores,  it  is  possible  to  achieve  the  sensitivity  that  is  comparable  to  the  one  obtained  with
isotopes  but  some  problems  could  occur  especially  when  competing  nucleic  acid  is  added  to  the
samples or if a ssDNA is the target. In such cases the fluorescent tag coupled to dNTP (usually dCTP,
dUTP)  could  be  a  solution.  Labeling  of  the  target  nucleic  acid  could  be  performed  during  a  PCR
reaction, by 3’-end filling with the Klenow fragment or by adding labeled nucleotides to the 3’-end by
the terminal deoxynucleotidyl transferase (TdT).
Another alternative to radioisotopes is chemiluminescent detection of the target nucleic acid, where
biotin or digoxigenin (DIG) can be used as the labels. Visualization of the target requires transfer of
the  labeled  molecules  to  a  solid  membrane  support  (nylon,  nitrocellulose)  and  subsequently,
a secondary detection method with the use of sterptavidin or anti-DIG antibodies. Since one biotin
can be bound by one streptavidin molecule with multiple covalently attached alkaline phosphatase
molecules, that can carry out many reactions with a lumigenic substrate, the detected signal could be
increased. The detection limit achieved for the biotin-streptavidin-alkaline phosphatase system could
be 5-50 pg of nucleic acid per band.

Binding conditions

The binding conditions such as salt concentration, pH or temperature should be adjusted in

case  of  a  particular  complex  formation.  Addition  of  substances  stabilizing  the  nucleoprotein
complexes  (glycerol,  sucrose,  proteases  or  nucleases  inhibitors)  or  reducing  the  nonspecific
interactions  (competitors,  non-ionic  detergents)  could  be  considered.  To  favor  the  specific
interactions between the protein and a nucleic acid the unlabeled competitors such as genomic DNA,
poly-d(I-C) or poly-d(A-T) are used. If the binding is specific, the addition of such competitors results
in the occurrence of distinct bands. If the competitor present in a sample reduces the target binding
it indicates non-specific interactions.

The typical volume of the reaction is 20-30 µl and an entire sample is usually loaded onto the

gel, however, the smaller volume of the loaded sample the narrower starting zone at the beginning
of separation and sharper bands at the end of electrophoresis.

Electrophoresis conditions

The conditions during electrophoresis have a big impact on the nucleoprotein complex

stability,  therefore  they  should  be  carefully  chosen.  The  most  commonly  used  buffers  are  Tris-
Borate-EDTA (TBE) and Tris-Acetate-EDTA (TAE) or their modifications if necessary (buffers without
EDTA for metal-binding proteins or without acid for increasing the complex stability). To stabilize the
complex during the electrophoresis, glycerol or ethylene glycol could also be added to the running
buffers.  If  the  nucleoprotein  interaction  is  extremely  weak,  the  binding  buffer  could  be  utilized
during the electrophoresis.

The sample separation could be performed in an agarose (0.7-1.5% w/v) or polyacrylamide

(3.5-6 % w/v) gel, however, a better resolution of the bands is obtained in case of the latter. Prior to
the preparation of the gels it is very important to degas all necessary solutions (TBE buffer,
acrylamide solution, H

O), what results in more efficient polymerization and in more homogenous

gels. When casting the gel, bubble formation should be also prevented, since bubbles do not conduct
electrical current what can affect the migration of the sample.

Before the separation of the sample in a gel, pre-electrophoresis, lasting 0,5-1h, should be

performed. It is a critical step as the gel is equilibrated with the running buffer and a use of a dye can

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

reveal any gel defects (gel homogeneity, shape of the lanes). The pre-electrophoresis, as well as the
actual separation, are conducted at 10 V/cm of gel length.

BRIDGED PROCEDURE

1.  Nucleic acid preparation and labeling.
2.  Preparation of the reaction mixtures containing nucleic acid titrated with a protein.
3.  Incubation of the mixtures in an appropriate temperature.
4.  Addition of a loading solution (glycerol/sucrose/ficoll).
5.  Loading the samples into the gel’s wells.
6.  Electrophoresis in an appropriate buffer.
7.  Detection of the electrophoretic bands (autoradiography, fluorescence imaging chemiluminescent imaging).

ROUBLESHOOTING

1. If no bands are visible after the electrophoresis :

−  the amount of the nucleic acid used should be verified and adjusted to the detection level;
−  the labeling reaction should be controlled and repeated if necessary;
−  nuclease inhibitors could be added to prevent nucleic acid degradation.

2. If no retardation is observed:

− the quantity and the activity of the protein should be verified;
− the migration of the protein alone should be verified; if it is the same as the migration of the target

nucleic acid the pH of the buffers should be reduced;

− the composition of the buffers should be verified (some proteins require very low salt concentration or

addition of metal ions).

3. If all bands are smeared:

−  fresh, degased reagents for gel preparation should be used;
−  the voltage could be reduced;
−  concentration of the gel and the running buffers should be checked.

4. If the free nucleic acid migrates normally and the retarded bands are smeared:

− conditions stabilizing the nucleoprotein complex should be used (minimal gel running time; increased gel

concentration; reduction of salt concentration in the buffers; lowering the electrophoresis temperature;
addition of stabilizing reagents to the buffers).

ITERATURE

Dahlberg A.E, Dingman C.W, Peacock A.C, 1969, Electrophoretic characterization of bacterial polyribosomes in

agarose-acrylamide composite gels, J Mol.Biol. 41:139-147

Fried M.G. and Crothers D.M, 1981, Equilibria and Kinetics of Lac Repressor-Operator Interactions by

Polyacrylamide Gel Electrophoresis. Nucl. Acids Res. 9:6505-6525

Hellman L.M. and Fried M.G, 2007, Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic

Acid Interactions, Nat. Protoc. 2(8):1849-1861

Lane D, Prentki P, Chandler M, 1992, Use of gel retardation to analyze protein-nucleic acid interactions,

Microbiol. Rev. 56(4):509-528

Rye H.S, Drees B.L, Nelson H.C, Glazer A.N, 1993, Stable fluorescent dye-DNA complexes in high sensitivity

detection of protein-DNA interactions, J Biol Chem 268(33):25229-25238

Travers A. and Buckle M, 2000, DNA-Protein Interactions, Oxford University Press

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 4. Footprinting

Among the many different methods enabling the investigation of nucleic acid – protein

interactions,  the  footprinting  technique  provides  knowledge  not  only  about  the  existence  of  such
complex formations but also enables to recognize the sequence bound by the protein. This method
was developed in the late 70s by Galaz and Schmitz, who, as an example, investigated interactions of
the  repressor  LacI  with  a  sequence  of  the  operator  lacO.  They  combined  two  techniques:  the
Maxam-Gilbert  DNA-sequencing  method  and  the  method  of  isolation  DNAase-protected  fragment
and developed the protecting assay, in which digestion of labeled nucleic acid is locally inhibited by
the  bound  protein.  The  cleavage  of  nucleic  acid  usually  is  random  and  optimized  to  cleave  every
molecule just once and in different positions, what results in a wide range of fragments different in
length. The nucleic acid is not cleaved in the region bound by the protein, therefore the same group
of digestion products cannot originate and on a denaturing polyacrylamide gel that is seen as a gap
named footprint (Fig.8).

Fig. 8. The scheme of footprinting assay. (A) An analysis of the protein binding site. The protein is incubated

with labeled DNA, then the DNA is digested and the obtained fragments are dissolved in a poliacrylamide gel.

The lack of the visualized bands correspond to the site occupied by the protein. (B) A control reaction without

the protein.

digestion

denaturation

digestion

denaturation

digestion

denaturation

digestion

denaturation

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Before every footprinting experiment the choice of the nucleic acid substrate, labeling method and
cleavage agent should be made.

Nucleic acid

The length of the nucleic acid fragment used in this experiment is limited by resolution of
polyacrylamide gels and usually is about 50-200 base pairs long. The fragment can be generated by
restriction digestion of a plasmid containing the sequence of interest or in a PCR reaction. The choice
of the nucleic acid is more complicated when the region bound by the protein is unknown and then
the application of artificial fragments could be useful.

Labeling method

Since in the footprinting assay very small amounts of nucleic acid is used (a few femtomoles of
nucleic acid), labeling with

P or

P is commonly applied. Fluorescent labeling (with fluorescein,

Alexa dyes, Texas Red, Cy-5) is also more and more popular since it is safer and the labeled fragment
does not decay. Unfortunately sometimes the sensitivity of the fluorescent technique is not enough
to observe bands containing less than 1% of total nucleic acid.
When the target is a restriction fragment, labeling with radioisotopes can be performed by 3’end
filling of the sticky-end with α-

P-dNTP by Klenow fragment. If the target is amplified during PCR

reaction, the radiolabeled at 5’end oligonucleotides can be used.

Cleavage agents

There are different agents used to cleave nucleic acid during the footprinting assay. The most

popular are DNaseI and hydroxyl radicals.

DNase I is an agent of choice if the nucleic acid is double-stranded DNA. It binds the DNA minor

groove  and  cuts  the  phosphodiester  backbone  of  the  molecule  in  a  divalent  cation-  dependent
manner. Both magnesium and calcium ions can be used, however the first ones are more efficient.
Requirement  of  divalent  ions  entails  the  simplicity  in  stopping  the  reaction  by  addition  of  EDTA.
DNaseI is also readily used in the footprinting assay since it is cheap and active in a wide range of
conditions. It can stand pH from 5 to 9, temperature up to 70°C and salt concentration even up to 1M
NaCl. The additional advantage of DNaseI is that it is sequence unspecific. Unfortunately depending
on  the  local  DNA  structure,  it  can  generate  an  uneven  range  of  digestion  products.  For  instance
the activity of the enzyme can be inhibited within AT-rich tracts because of narrower minor groove
hindering the protein binding. Also the GT-rich regions are not preferred by DNaseI because of lower
flexibility. Easiness of DNA bending by the enzyme seems to be a required element of the DNaseI
action mechanism. The disadvantage of this enzyme is also its size, because as a large glycoprotein
that binds 10 base pairs, it overestimates the protein binding sequence by about 3 base pairs.

The second commonly used cleavage agent are hydroxyl radicals. They are generated in

a Fenton reaction, during which the Fe

is oxidized to Fe

by hydrogen peroxide. The products of

this reaction are also hydroxyl radical and hydroxyl anion. Since the Fe

can interact with negatively

charged DNA, in practice ferric ion used in the reaction is chelated by EDTA ([Fe(EDTA)

]). In the

reaction sodium ascorbate is added, since it reduces the [Fe(EDTA)

] product back to [Fe(EDTA)

what provides the starting amount of the Fenton reaction substrate. Generated hydroxyl radicals are
highly reactive and attack the C4’ or C1’ positions of deoxyribose via the minor groove of the DNA.
Because of their small size, they are able to cleave very close to the bound protein. The cleavage is
sequence unspecific, however it can be reduced within the AT tracts. The cleavage reaction is slower

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

than with DNaseI, sensitive to solvents scavenging free radicals (DMSO, ethanol, glycerol) and
requires precipitation of reaction products before polyacrylamide gel electrophoresis. The reaction is
quenched by addition of a thiourea stop solution, which reacts with any remaining hydroxyl radical.

Electrophoresis conditions

The samples are resolved in a denaturing polyacrylamide gel, where the denaturing agent is

7M  urea  and  the  running  buffer  is  usually  TBE.  The  polyacrylamide  concentration  within  the  gel
varies from 5 to 20% depending on the DNA fragment size (8% gel is a standard for 150bp fragment)
and  the  prepared  gel  is  very  thin  (0.33mm)  and  long  (40cm).  The  samples  should  be  heated  and
quickly cooled on ice before loading onto a gel to enable denaturation of the cleavage products.

BRIDGED PROCEDURE

1.  DNA fragment preparation (PCR reaction with labeled primers; restriction digestion)
2.  Labeling of DNA fragment (unless the fragment was labeled during PCR reaction)
3.  Binding reaction (the condition of the reaction should be adjusted)
4.  Cleavage of DNA (the type, amount of cleavage agent and the reaction condition should be adjusted)
5.  Precipitation of cleavage products (it is very important when the hydroxyl radicals are the cleavage agents;

when DNaseI is used there is usually no need to remove the enzyme)

6. Denaturing polyacrylamide gel electrophoresis
7. Detection of electrophoretic bands (autoradiography, fluorescence imaging)

Modifications of the method

Solid-phase footpriniting

In this method the labeled DNA fragment is immobilized via biotin–streptavidin interaction on

paramagnetic beads. Then the protein of interest is added and the nucleoprotein complex is formed.
The next step is cleavage of DNA by a cleavage agent (usually DNaseI) and purification of nicked DNA.
Since the nucleic acid is immobilized on solid phase, the purification of DNA is facilitated what results
in a faster protocol. After the digestion, the resin is washed and samples are denatured. The beads
can be easily removed by simple centrifugation and the DNA of interest remains in the supernatant.
The separation of cleavage products is performed as in the classic footprinting method.

Nucleic acid cleavage and primer extension

Although DNaseI and hydroxyl radicals have a lot of advantages, sometimes other cleavage

agents are used as nucleases mung bean, P1 and S1. They are agents of choice when the nucleic acid
is  single  stranded  DNA  or  RNA.  The  nucleoprotein  complex  formation  followed  by  nucleic  acid
degradation by these agents is the same as in the classic footprinting assay. The difference is that the
target nucleic acid is not labeled and the cleavage is followed by primer extension assay, in which
a labeled primer is used (Fig. 9). The cleavage agent is used in a concentration, which enables to cut
every molecule of the nucleic acid just once. The bound protein protects the potential cleavage sites
what  is  observed  as  the  absence  of  a  nucleic  acid  fragment  of  a  particular  length.  Because
the products  of  the  cleavage  reaction  are  single  stranded  nucleic  acid  fragments,  to  facilitate
the visualization  of  the  reaction  products  a  one  labeled  primer  is  used.  In  the  PCR  reaction
the complementary strands are synthesized and a ladder of double stranded fragments is produced.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

As a control, the reaction without protein is prepared. The comparison of primer extension products
of these two reactions allows to indicate the nucleic acid region bound by the protein.

Fig. 9. The scheme of the footprinting assay

with the primer extension reaction. (A) An

analysis of protein binding site. The protein

is incubated with non-labeled DNA, then the

DNA is digested and the obtained fragments

are used as a template for the PCR reaction

with a labeled primer. Then the reaction

products are dissolved in a poliacrylamide

gel and the lack of the visualized bands

correspond to the site occupied by the

protein. (B) A control reaction without the

protein.

In vivo footprinting

The classical footprinting assay required the interaction of purified protein with prepared

nucleic  acid  fragment.  Unfortunately  sometimes  the  reconstruction  of  native  conditions  is  very
difficult and the lack of complex formation does not exclude the formation of such a complex in vivo.
The results obtained from live cells can give more reliable information. However, in case of in vivo
studies, the exposition of cells to the action of the cleavage agents for several minutes can result
with  artificial  results.  It  is  mainly  because  the  used  chemicals  can  interact  with  many  cell
components.  These  kinds  of  limitations  can  be  avoided  when  UV-light  is  a  footprinting  agent.
The irradiation  of  DNA  results  with  a  wide  range  of  photoproducts  depending  on  local  DNA
environment and the assumption is that one molecule is photomodified just once. The most frequent
photoproducts formed in DNA are pyrimidine dimers, purine dimers are less frequent. The reactivity
pattern is different for necked nucleic acid and molecule bound by the protein. After the irradiation
the  nucleic  acid  is  isolated  from  the  cells  and  primer  extension  reaction  with  labeled  primer  is
performed.  The  polymerase  stops when  it meets  the  photo-changed  nucleotide  and  the  ladder of
amplification  products  is  generated.  They  are  separated  on  a  denaturing  polyacrylamide  gel  and
detected depending on the labeling method.

Potassium permanganate probing assay

The potassium permanganate (KMnO

) probing assay is not a typical technique for detection of

protein interactions with DNA. It is usually used to observe local melting of dsDNA and formation of
a ssDNA region. However because of the similarities to the footprinting method it is often called
“KMnO

footprinting”. Compared with nucleases, KMnO

is an oxidizing agent that modifies thymine

residues in single stranded DNA. The agent concentration is adjusted to obtain just one modification
per molecule. This reagent does not modify the residues in double stranded nucleic acid. The probing
with KMnO

is followed by primer extension with a labeled primer (see above). During this reaction,

the polymerase stalls at the modified thymines on the template, what results with a range of

denaturation

primer
extension

denaturation

primer
extension

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

products of different length. The assay with KMnO

probing is typically use to identify a region within

the nucleic acid where the local melting of double stranded molecule and formation of a single
stranded region occurs (“opening”).

ROUBLESHOOTING

1. If no or low cleavage of nucleic acid is observed:

− check the cleavage agent (the DNaseI concentration and activity; if hydroxyl radicals are used prepare

new reagents)

− check the condition of the reaction, if there are no inhibitors of the cleavage agent

2. If the cleavage is observed within the expected footprint region:

−  lower the concentration of cleavage agent
−  reduce the time of cleavage reaction
−  check the conditions of the binding reaction
−  check the concentration and activity of the protein

3. If all bands are smeared:

− fresh, degassed reagents for gel preparation should be used
− the concentration of gel and running buffers should be checked

ITERATURE

Hampshire A. J, Reusling D. A, Broughton-Head V. J, Fox K. R, 2007, Footprinting-A method for determining the

sequence selectivity, affinity and kinetics of DNA-binding ligands, Methods 42: 128-140.

Pfeifer G.P. and Tornaletti S, 1997, Footprinting with UV Irradiation and LMPCR, Methods 11: 189-196.
Sandaltzopoulos R. and Becker P. B, 1995, Solid-phase DNaseI footprinting, Biochemica 4: 25-27.
Travers A. and Buckle M, 2000, DNA-Protein Interactions, Oxford University Press.

UESTIONS

1.  What cleavage agents can be used in a footprinting experiment?
2.  Can the footprinting assay be an in vivo experiment?
3.  What type of footprinting method would you use for detection of:

a. protein-DNA interaction sites
b. dsDNA melting regions
c. nucleoprotein interactions within Adenine and Thymine rich regions?

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 5. Surface Plasmon Resonance (SPR)

One of the most sophisticated techniques enabling investigation of the interactions between

molecules  in  real  time  is  the  surface  plasmon  resonance  (SPR).  It  is  a  specific  chromatography
technique since two phases are present in the experiment: the mobile phase (analyte) passes over
the  immobile  phase  (ligand  attached  to  the  surface  of  the  sensorchip).  In  SPR  technique  a  very
sensitive  detection  system  is  used,  what  makes  performing  experiments  with  a  trace  amount  of
substance  and  observing  even  cryptic  interactions  possible.  Moreover,  the  use  of  the  available
software gives the possibility to calculate the values of constants from the obtained results. The first
use  of  the  SPR  concerned  the  immuno-complex  formation.  However,  the  development  of  this
method  nowadays  enables  analyzing  interactions  between  different  molecules,  like  nucleic  acids,
proteins, lipids or even the entire cells and viruses. The SPR is a powerful technique applied not only
in life sciences but also in drug discovery or food analysis.

Fig. 10. The schematic SPR de-

tection system. The basic

elements such as prism, sensor

chip and a flow cell are marked

at the scheme. The black arrow

shows the direction of the flow

through the flow cell.

The physical basis of SPR is an optical method that enables the measurement of the angle of

light  reflected  from  the  surface  of  a  sensor  chip  coated  with  a  thin  golden  film  (Fig. 10).
The reflection of the incident polarized light is not total because some of the energy is lost in the
film.  When the  angle of the  incident  light  is  proper,  it  can  cause  a  resonance  of mobile  electrons
(plasmons) present at the surface of the gold layer. The resonance frequency and indirectly the angle
of the reflected light depend on the refractive index, i.e. the medium surrounding the sensor chip
surface.  Contact  with  a  buffer  or  binding  a  ligand  to  the  surface  of  a  sensor  chip  changes
the refractive  index  and  therefore  the  reflection  angle.  This  angle  is  called  surface  plasmon
resonance angle and the greater the mass immobilized on the surface of the sensor chip the greater
changes in the SPR angle detected. The changes in the spr angle are reported as response units (RU)
and  every  1000  RU  corresponds  to  about  0.1°  change  in  the  reflection  angle,  which  is  caused  by
binding approximately 1 ng of the protein on every mm

of the sensor chip.

Nomenclature

Ligand – a molecule attached to the surface of the sensor chip
Analyte – a molecule that is passed in the solution over the immobilized ligand
Sensogram – a plot of the response against time, showing the progress of the interaction

prism

incident

light

reflected

light

flow cell

sensor chip
(glas sidle,

gold layer)

ligand

analyte

SPR angle

prism

incident

light

reflected

light

flow cell

sensor chip
(glas sidle,

gold layer)

ligand

analyte

SPR angle

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Sensor chips

The sensor chip is a glass surface, that is coated with a very thin layer of gold. Usually

the metal layer is modified and a dextran hydrogel layer is added to form a hydrophilic environment
on  the  surface  of  the  sensor  chip.  This  modification,  and  also  some  others,  facilitate
the immobilization  of  the  molecules.  One  sensor  chip  contains  4  flow  cells  (channels),  what  gives
a possibility  to  perform  3  experiments  simultaneously.  The  fourth  channel  remains  empty
and functions as a reference one.
Depending on the sensor chip surface character, we can distinguish:
−  CM1 - contains a carboxymethylated surface without dextran; has low binding capacity; is suitable

for binding large molecules, viruses, entire cells; couples molecules to carboxyl groups on the
sensor surface via -NH

, -SH, -CHO, -OH or –COOH groups

− CM3 - contains a surface with carboxymethylated short dextran chains; it is suitable for binding

large molecules, viruses, entire cells; couples molecules to carboxyl groups on the sensor surface
via -NH

, -SH, -CHO, -OH or –COOH groups

− CM4 - contains a surface with dextran chains as long as in the CM5 sensor chip (longer than in

CM3) but with lower carboxymethylation; due to a reduced negative charge of the surface it is
suitable for immobilization of negatively charged molecules

− CM5 - one of the most commonly used sensor chips; contains a surface with carboxymethyleded

dextran chains longer than in the CM3 chip; suitable for binding small to large molecules or whole
viruses; couples molecules to the carboxyl groups on the sensor surface via -NH

, -SH, -CHO, -OH

or –COOH groups

− CM7 - similar to the CM5 sensor chip but contains a surface with higher carboxymethylation of

the dextran chains and higher density of the matrix, what provides three times higher
immobilization capacity; suitable for binding small molecules; couples molecules to the carboxyl
groups on the sensor surface via -NH

, -SH, -CHO, -OH or –COOH groups

− NTA - contains a surface with carboxymethylated dextran chains, pre-immobilized with

nitrilotriacetic acid (NTA); suitable for binding His-tagged proteins

− SA - contains a surface with carboxymethylated dextran chains, pre-immobilized with

streptavidin; suitable for binding molecules tagged with biotin

− L1 - contains a surface with carboxymethylated dextran with covalently attached lipophilic groups;

suitable for binding lipid membrane vesicles; provides the maintenance of the lipid bilayer
structure

− HPA - contains a hydrophobic surface with a long-chain of alkanethiol molecules, attached directly

to the golden film; suitable for binding lipid monolayers

Immobilization methods

Every SPR analysis begins with immobilization of a ligand on the surface of the sensor chip.

There are three main methods enabling to attach molecules: covalent immobilization, immobilization
via coupled molecule or hydrophobic adsorption. The chosen method depends on the character of
the immobilized molecule and on the type of the sensor chip, which is going to be used.

Covalent immobilization is the most popular method used to attach molecules on the surface

of the sensor chip. The CM sensor chips are suitable for this method and the CM5 is usually the chip

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

of  first  choice.  It  requires  modification  of  the  ligand  and  provides  its  stable  attachment  in
the conditions used in the course of an experiment. The conditions of the immobilization reaction
should take into consideration: the temperature (usually 25°C), pH (3,5<pH<pI of the protein) and the
buffer composition (e.g. for amine coupling Tris should be omitted). Before the attachment of the
molecules  the  surface  of  the  sensor  chip  requires  activation.  Addition  of  1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide  (EDC)  and  N-hydroxysuccinimide  (NHS)  to  the  carboxy-
methylated  matrix  of  the  chip  results  in  the  formation  of  succinimide  esters,  that  spontaneously
react  with  a  ligand.  Attaching  the  ligand  can  utilize  three  coupling  chemistries:  via  amine  (amine
coupling), thiol (thiol coupling) or aldehyde (aldehyde coupling) groups. After the immobilization of
the  ligand,  the  excess  of  the  reactive  group  should  be  inactivated  by  addition  of  an  appropriate
chemical, for instance ethanolamine in case of the amine coupling. It should be remembered that in
this method it is difficult to determine the orientation of the bound ligand. The most heterogeneous
orientation  is  given  by  the  amine  coupling  since  most  proteins  contain  several  available  amine
groups.

The defined attachment orientation can be provided by the capturing approach. It utilizes

the immobilization  of  the  ligand  via  a  capturing  molecule,  covalently  attached  to  the  surface  of
the sensor chip. Streptavidin (sensor chip SA) or NTA (sensor chip NTA) can be such molecules and
they require that the ligand contains biotin or His-tag, respectively. The surface of the sensor chip
can  also  be  prepared  individually  and  other  tags  or  antibodies  can  be  used  as  the  capturing
molecules. Unfortunately, the maximum analyte binding capacity is usually lower when compared to
a directly immobilized ligand.

The third method of the immobilization of the molecules is a hydrophobic attachment based

on lipid monolayers (sensor chip HPA) and bilayers (sensor chip L1). The lipid monolayer is created by
adsorption of lipids from micelles or liposomes on the surface of the sensor chip. They are bound in
a way that the hydrophobic lipid tails are oriented towards the golden film and the hydrophilic heads
towards  the  aqueous  sample.  In  case  of  creating  a  sensor  chip  with  a  lipid  bilayer,  the  chip  with
a matrix  containing  a  dextran  with  hydrophobic  structures  can  be  used.  These  structures  can  be
inserted into the liposomes and attach the membranes to the dextran matrix.

When the method of immobilization is chosen, a proper amount of ligand that is going to be

attached should be estimated. The amount of the bound ligand is expressed as ligand binding
capacity presented in response units RU. It depends on the molecular weight of both the ligand and
the analyte and can be calculated from the formula:

The SPR experiment

When a ligand is immobilized on the surface of the sensor chip, the actual experiment can be

performed. Three phases can be distinguished during a SPR experiment: association, dissociation and
regeneration (Fig. 11). In the first step an analyte dissolved in a suitable buffer (called running buffer)
passes over the immobilized ligand and interacts with it. If the two form a complex the mass
attached to the gold layer rises, what changes the spr angle and causes an increase of the detected
signal. The second phase is dissociation, when the analyte is omitted from the running buffer and the
formed complex dissociates. During this phase a detected response decreases. In the last stage of

ligand binding

capacity (RU)

analyte binding

capacity (RU)

ligand molecular

weight

analyte molecular

weight

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

the experiment,  the  surface  of  the  sensor  chip  is  regenerated  by  removing  the  remnants  of
the analyte.  What  is  important,  the  ligand  is  not  damaged  during  this  phase  and  after
the regeneration the next experiment can be performed. However, it should be remembered that
the surface of the sensor chip with the immobilized ligand is not indestructible and approximately
after 40 analyte injections a noticeable decrease in the analyte binding capacity is detected. In this
situation the sensor chip should be changed.

Fig. 11. Schematic illustration of a sensogram. The three main phases of the spr experiment (1-3) and response

points (a, b) important to calculate analyte binding response (relative response; c) are marked on the diagram.

BRIDGED PROCEDURE

1.  Activation of the surface of the sensor chip and preparation of the ligand
2.  Immobilization of the ligand
3.  Injection of the analyte
4.  Analysis of the interaction
5.  Regeneration of the surface of the sensor chip

ROUBLESHOOTING

1. If no association of an analyte, expected to bind the ligand, is detected:

−  verify the binding conditions; when required, change the buffer
−  verify the analyte activity
−  verify the analyte concentration

2. If the signal detected on the reference channel increases

− use more stringent experimental conditions
− change the sensor chip

3. If the analyte binding capacity decreases

− verify the analyte activity
− change the sensor chip

association

dissociation

regeneration

baseline

response (RU)

relative
response (RU)

Time (s)

(

)

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

ITERATURE

Biacore Sensor Surface Handbook BR-1005-71 Edition AB.
Cullen DC, Brown RG, Lowe CR, 1987-1988, Detection of immuno-complex formation via surface plasmon

resonance on gold-coated diffraction gratings. Biosensors 3(4): 211-25.

Schasfoort R.B.M. and Tudos A.J, 2008, Handbook of Surface Plasmon Resonance, The Royal Society of

Chemistry Publishing.

UESTIONS

1. What kind of sensorchip would you use during analysis of protein interactions with

a. DNA
b. proteins

2. Describe the bases of ligand attachment by the capturing approach.
3. The figure below presents sensograms of proteins A (A) and B (B) binding to the DNA fragment, immobilized

on the surface of a SA sensorchip. The molecular mass of protein A is 50kDa and protein B 100kDa. What
can you say about the interaction of the proteins A and B with the analyzed DNA fragment?

Time (s)

(RU

)

500

250

375

125

150

250

350

450

Time (s)

(RU

)

500

250

375

125

150

250

350

450

Time (s)

(RU

)

500

250

375

125

150

250

350

450

Time (s)

(RU

)

500

250

375

125

150

250

350

450

Time (s)

(RU

)

500

250

375

125

150

250

350

450

Time (s)

(RU

)

500

250

375

125

150

250

350

450

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 6. Chromatin immunoprecipitation assay (ChIP)

Among the many techniques that enable investigation of interactions between proteins and

nucleic  acids,  the  chromatin  immunprecipitation  assay  (ChIP)  is  utilized  for  detection  of
a nucleoprotein  complex  formation  in vivo.  The  method  is  based  on  fixation  (crosslinking)  of
nucleoprotein complexes within cells at any point of the cell cycle, fragmentation of nucleic acid and
isolation  of  a  protein-nucleic  acid  complex  via  specific  antibodies  that  recognize  the  protein  of
interest (Fig. 12). In the original method the DNA isolated from the complex is used as a template in
a PCR reaction (see Chapter 1). There are also experiments called “RIP” where it is the RNA that is
co-precipitated with a protein and then it is submitted to a reverse transcription reaction.

Fig. 12. A scheme of the Chromatin Immunoprecipitation

assay (ChIP). The protein of interest is marked as a red

circle, the protein A from Staphyclococcus aureus is

marked as a gray circle and the black ellipse represents

sepharose beads. The scheme presents the basic steps of

the ChIP assay: Crosslinking of nucleoprotein complexes;

Cell lysis and nucleic acid fragmentation; Incubation with

antibodies; Precipitation; Nucleic acid purification.

The ChIP technique was used mainly to determine the binding sites of transcription factors and

histone  proteins.  Now  it  is  applied  to  recognize  binding  sites  for  many  other  proteins  and  is
combined  with  other  techniques,  such  as  real-time  PCR,  microarrays  (ChIP-on-chip),  cloning  or
sequencing (ChIP-seq).

Precipitation

Crosslinking of nucleoprotein complexes

Cell lysis and nucleic acid fragmentation

Incubation with antibodies

Decrossling and nucleic acid purification

sepharose

Detection e.g. PCR

Precipitation

Crosslinking of nucleoprotein complexes

Cell lysis and nucleic acid fragmentation

Incubation with antibodies

Decrossling and nucleic acid purification

sepharose

Detection e.g. PCR

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Although the procedure is simple, optimization of conditions is usually needed. The optimal

cross-linking agent, fixation conditions, nucleic acid fragmentation method, immunoprecipitation
conditions and post-immunoprecipitation detection technique should be carefully chosen.

Cross-linking agent

Fixation of cellular structures can be obtained by chemical (acridine orange, cisplatin,

formaldehyde, methylene blue) or UV light crosslinking.

Formaldehyde is the most popular crosslinking agent since the fixation is very simple and it can

be easily reversed. Formaldehyde can be added directly to the cell culture because it penetrates the
cell membranes and crosslinks proteins to nucleic acids. In a protein it reacts with amine and imine
groups of amino acids such as lysines, arginines, histidines and in a nucleic acid with the side chains
of adenines and cytosines, what results in the formation of Shiff’s bases. The time of incubation with
formaldehyde  should  always  be  adjusted  individually  since,  for  example,  non-histone  proteins
require longer incubation. However, it should be taken into consideration that longer exposition of
cells to this chemical can also cause protein-protein crosslinking, what in consequence can give false-
positive  result  or  it  can  hinter  nucleic  acid  fragmentation  leading  to  false  negative  results.
Formaldehyde  is  also  a  mild  denaturing  agent  and  during  an  exposition,  which  is  too  long,
denaturation of proteins can occur, what can decrease the antigen availability. Therefore the fixation
time  should  not  be  exceeded  and  on  average  should  be  up  to  10-30  minutes.  The  crosslinking
reaction can be stopped by the addition of glycine solution (0.125 M) and reversed by heating (6h
at 65°C).

Other crosslinking chemicals are less often used, since they could be very toxic or crosslink

only DNA with proteins, like for example cisplatin. Some of them (acridine orange, methylene blue)
are agents of choice if nucleases or restriction enzymes are to be used for fragmentation of nucleic
acids, since after the formaldehyde fixation only sonication can be applied.

Besides the use of chemical crosslinking agents it is also possible to utilize ultraviolet light for

cells fixation. The source of light should be in a distance of 2-3 cm from a dish with the cell culture.
The cell irradiation with 254 nm or 266 nm UV light takes usually from a few seconds (for laser as the
source  of  light)  up  to  15  minutes  (for  low  intensity  light  source)  and  crosslinks  nucleic  acids  with
proteins.  The  first  crosslink  was  shown  for  cysteine  with  uracyl  and  cysteine  with  thymine.  Apart
from  the  cysteine,  the  most  photoreactive  amino  acids  are  phenylalanine  and  tyrosine.
Unfortunately, over-irradiation can cause damage both in proteins and nucleic acids and can bring
false negative results. Appropriate irradiation time should be adjusted in every experiment.

Nucleic acid fragmentation method

To facilitate the purification of a nucleoprotein complex from the cell lysate and to enable

reliable estimation of the region of nucleic acid bound by the protein, fragmentation of nucleic acid is
required. The most commonly used method is sonication, which utilizes ultrasounds for shredding
nucleic acids, what produces fragments from 100 to 500 bp in length. Usually several cycles, a few
seconds burst and a few seconds pause, are applied. It is very important to keep samples chilled on
ice during the sonication since heat released during this process can result in denaturation of nucleic
acids and proteins. After the nucleic acid fragmentation with ultrasounds the sample can be
immediately subjected to immunoprecipitation.

An alternative for the sonication method is digestion of the nucleic acids with nucleases. It is

often used when no crosslinking agent was added or after a UV-light crosslinking. The reaction is fast

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

and simple, the incubation is performed in 37°C for 5 min. It can be easily stopped by addition of
EDTA, that chelates divalent ions, which are cofactors for nucleases. Digestion with nucleases is not
recommended after formaldehyde crosslinking because due to the nucleic acid fixation the cleavage
is not efficient. A disadvantage of this method is the necessity of purification of nucleic acid
fragments after the cleavage reaction. The procedure requires centrifugation of the reaction mixture
in a CsCl gradient, which is time-consuming.

Immunoprecipitation conditions

One of the most important steps during the whole ChIP procedure is immunoprecipitation of

the  nucleoprotein  complex  from  a  cell  lysate.  It  is  performed  by  addition  of  specific  antibodies
recognizing  the  protein  of  interest  bound  to  the  nucleic  acid  and  subsequent  isolation  of  such
a complex.  Concentration  of  the  antibodies  and  the  time  of  incubation  depend  on  the  antibody
specificity  and  should  be  adjusted  experimentally.  After  the  recognition  of  the  nucleoprotein
complex  by  the  antibodies,  the  whole  complex  is  purified  by  utilization  of  protein  A  from
Staphyclococcus aureus or protein G from Streptococcal bacteria, conjugated with sepharose beads.
These proteins are able to bind with high affinity to the Fc region of an antibody. Conjugation with
the sepharose beads facilitates the isolation of such a complex by simple centrifugation. Although
the method of purification of the immunoprecipitated complex is quite fast and easy, a problem with
high background can occur. It arises from unspecific interactions of proteins and nucleic acids with
sepharose beads. To avoid it, competitors such as BSA or sonicated salmon sperm DNA are added to
the  samples.  Also  the  use  of  magnetic  beads  can  solve  this  problem,  however,  it  is  still  quite
an expensive technique.

Post-immunoprecipitation detection methods

After the isolation of the nucleoprotein complex through the interaction with antibodies and

the proteinA-sepharose beads, the nucleic acid should be isolated for further analysis. If the DNA is
the nucleic acid and formaldehyde is the crosslinking agent, it can be achieved by a simple incubation
of the sample in a TE buffer at 65°C for 6h, followed by centrifugation. The supernatant contains the
DNA fragments released from the complexes. Also, the whole nucleoprotein complex can be released
during incubation with 1% SDS and 0,1 M NaHCO

solution, for 15 min at room temperature. Then

the protein-DNA complex is further incubated at 65°C for 6h, to de-crosslink (reverse crosslink) and
release the nucleic acid. Addition of proteinase K helps to degrade proteins present in the sample.
The isolated nucleic acid can be further analyzed in a PCR reaction, Southern blotting or sequencing
reaction.

In case of RNA-protein complexes the procedure is very similar. After the isolation of

the complex with antibodies and beads coated with the protein A, an elution buffer (100 mM Tris, 10
mM EDTA, 1% SDS and DEPC-H

O) is added and the sample is incubated for 10 minutes at 37°C,

followed by centrifugation. The supernatant contains the RNA-protein complexes, from which pure
RNA can be obtained by proteinase K-mediated degradation of the protein. Subsequently the RNA
can be utilized in a RT-PCR or northern blotting analysis.

If a protein is going to be analyzed, the Laemmli buffer can be added directly to the sample

containing beads with isolated complexes. Next, after heating and centrifugation the supernatant can
be loaded onto denaturing polyacrylamide gels and SDS-PAGE followed by western blotting can be
performed.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Control experiments

When preparing a ChIP analysis one should carefully plan control experiments, since difficulties

at every step of the procedure can give false results. A sample of the cell lysate should be taken to
confirm the presence of the nucleic acids and proteins in the initial material. To exclude over-fixation,
cross-reactivity of antibodies or unspecific interactions with resin, preparation of samples omitting
the addition of the crosslinking agent, antibodies or protein A-sepharose beads, should be
performed. If it is available, a lysate from cells with the deletion of a gene encoding the protein of
interest could also be used as a negative control.

BRIDGED PROCEDURE

1.  Crosslinking of nucleoprotein complexes within the living cells (chemicals, UV-light)
2.  Lysis of the cells (enzymatic; mechanical; ultrasounds)
3.  Nucleic acid fragmentation (sonication; enzymatic cleavage)
4.  Incubation with antibodies specific for the protein of interest
5.  Incubation with beads coated with protein A or protein G
6.  Purification of the complexes by centrifugation
7.  Isolation of nucleic acid-protein complexes, necked nucleic acid or protein
8.  Further analysis (PCR, blotting, sequencing)

ChIP and other methods

ChIP-chip

One of the most popular modifications of the ChIP technique is the so called ChIP-on-chip,

which  combines  classical  the  immunoprecipitation  assay  with  microarrays.  It  enables  very  fast
screening of whole genomes for regions bound by proteins of interest. For example, the combination
of these two methods was successfully applied for the detection of regions bound by transcription
factors in yeasts. The method for obtaining the nucleic acid fragments in the ChIP-chip is the same as
in  the  classic  immunoprecipitation  assay.  The  additional  steps  are  the  fluorescent  labeling  of
the obtained fragments and hybridization to a genome microarray. As a control of the background,
the initial nucleic acid fragments (after sonication) labeled with a different dye can be used. The most
popular dyes utilized in this technique are Cy3 and Cy5.

ChIP cloning

In a ChIP-cloning method the nucleic acid fragments obtained from the ChIP assay are cloned

into vectors. This results in the construction of a library, containing fragments bound by a particular
protein. In this technique it is very important to obtain as low a background as possible since nucleic
acid fragments bound nonspecifically by the protein have equal chance to be cloned as
the specifically-bound fragments.

ChIP-seq

To identify an unknown sequence recognized by the protein of interest, the ChIP assay

followed by a sequencing reaction can be performed. It is usually utilized for creating binding maps
of whole genomes and requires analysis of large packages of data. There are many software tools
and sophisticated algorithms that assist during such data analysis and help disprove false results.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

ROUBLESHOOTING

1. If no nucleic acid is detected after ChIP procedure:

− verify the initial nucleic acid content (if necessary, add nuclease inhibitors; increase the number of cells

used in the experiment);

− verify the conditions of the crosslinking reaction (formaldehyde can cause over-fixation and masking of

the nucleic acid by proteins; UV-light can damage both nucleic acid and the protein);

− verify the presence of the initial protein (if necessary add the protease inhibitors);
− check the specificity of antibodies (if necessary, adjust the concentration and time of incubation with

antibodies).

2. If nucleic acid is detected in a negative control reaction:

−  add competitors (BSA, salmon sperm DNA);
−  reduce the time of incubation with a resin;
−  increase the concentration of a salt in the sample;
−  add a detergent (TritonX, NP40, Brij 58).

ITERATURE

Buck M.J. and Lieb J.D, 2004, ChIP-chip: considerations for the design, analysis, and application of genome-

wide chromatin immunoprecipitation experiments, Genomics 83: 349-360.

Das P.M, Ramachandran K, vanWert J, Singal R, 2004, chromatin immunoprecipitation assay, BioTechniques

37(6): 961-969.

Smith K.C, Aplin R.T, 1966, A mixed photoproduct of uracil and cysteine (5-S-cysteine-6-hydrouracil). A possible

model for the in vivo crosslinking of deoxyribonucleic acid and protein by ultraviolet light, Biochemistry

5:2125-2130.

Smith KC, 1969. Photochemical addition of amino acids to 14C-uracil, Biochem. Biophys. Res. Commun. 34: 354-

357.

Smith K.C, 1970, A mixed photoproduct of thymine and cysteine: 5-S-cysteine, 6-hydrothymine. Biochem.

Biophys. Res. Commun, 39: 1011-1016.

Travers A. and Buckle M, 2000, DNA-Protein Interactions, Oxford University Press.

UESTIONS

1.  Is it possible to identify a sequence of a DNA fragment bound by the protein, analysed by ChIP?
2.  What modifications of the ChIP technique increase output of the method?
3.  Protein X has been analyzed for its binding to DNA fragment by ChIP. How would you interpret the obtained

result presented on the figure below? What kind of additional control(s) would you propose?

PCR product

1 2 3 4

ells

lin

ells

lin

ells

lin

ont

rol

tion

PCR product

1 2 3 4

ells

lin

ells

lin

ells

lin

ont

rol

tion

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 7. Microscopy

Researchers’ adventure with microscopy began in the XVII century, when Leeuwenhoek

improved  the  construction  of  the  microscope  and  was  first  to  start  observing  living  cells.
The microscope  he  was  using  was  a  light  microscope  and  since  those  days  it  was  improved many
times  resulting  in  a  wide  range  of  different  instruments  that  enable  observations  of  subcellular
structures.  One  of  the  greatest  breakpoints  took  place  at  the  beginning  of  the  XX  century  and
involved  the  utilization  of  beams  of  electrons  for  creating  a  specimen’s  image.  It  enabled  the
observation of molecules as small as the nucleic acids, proteins or nucleoprotein complexes. Then
the subsequent progress in microscopic imaging lead to the observation of molecules with atomic
resolution that allows to notice even subtle changes in a molecule’s structure.

Fluorescence microscopy

Light microscopy was the first technique developed to observe biological objects.

Unfortunately,  it  was  very  difficult  to  observe  molecules  as  small  as  nucleic  acids,  since  this
technique provides resolution of about 0,2 µm and results in good images only for dark or strongly
refracting  specimens.  For  better  imaging  of  biological  structures  fluorescence  microscopy  was
developed. It is also a type of light microscopy but the object of interest is illuminated with a nearly
monochromatic light that excites a specimen’s fluorescence. Then the emitted light passes through
an emission filter to the microscope lens. (Fig. 13) The presence of such a filter ensures that only
emitted light is going to be observed. Depending on the light source, also an excitation filter can be
used that provides monochromatization of the emergent light. Besides a source of light emission and
an excitation filter often there is also a dichroic mirror installed. It reflects the excitatory light beam
and divides it into a number of spectrally distinct output beams, what results in an illumination of the
specimen with an intense light.

Fig. 13. A scheme of light fluorescent

microscope. The elements of the

microscope such as ocular, objective,

emission and excitation filters, source of

light and dichroic mirror are marked on

the picture. The black arrow indicates

the whole light spectrum produced by

the lamp. The dark gray arrow shows the

path of the excitation light and the light

gray arrow represents the light emitted

by the specimen.

Fluorescence microscopy allows to observe a molecule that emits light by itself or that was

stained or tagged with a fluorescent molecule. The most popular dye utilized for DNA staining is DAPI
(

Light source

Ocular

Dic

oic

rro

tio

filt

Specimen

Emission filter

Objective

Light source

Ocular

Dic

oic

rro

tio

filt

Specimen

Emission filter

Objective

4',6-diamidino-2-phenylindole). It associates with AT clusters in the minor groove and preferentially

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

stains dsDNA. DAPI can also bind RNA but in a different binding mode. It is excited by a

Apart from dyes, naturally occurring proteins like green fluorescent protein (GFP), can be used

as fluorescent tags facilitating microscopic observations of nucleic acids and proteins. A gene for such
a tag can be fused with the gene for a protein, that binds the analyzed nucleic acid. Binding of
the product of this gene (fusion protein) to the nucleic acid can result in focusing of fluorescence in
a subcellular position, that corresponds to the subcellular position of the nucleic acid. This kind of
nucleic acid tagging is utilized for example in the fluorescent repressor-operator system (FROS).

wavelength of

358 nm and emits light of 460 nm wavelength (when bound with RNA it emits light of about 500 nm)
which is visible as blue fluorescence.

Fluorescent Repressor-Operator System (FROS)

The FROS technique enables localization of a DNA particle in living cells and is based on

the interaction  between  a  fusion  protein  (repressor  fused  with  a  fluorescent  protein)  and
an operator sequence introduced into the nucleic acid of interest. Binding of the operator sequence
by the fusion protein, via an interaction with the repressor, results in a convergence of fluorescence
in  the  position  occupied  by  the  nucleic  acid  (Fig.14).  Under  a  microscope  it  is  observed  as
a fluorescent focus.

Fig. 14. A scheme of FROS system based on lactose (A) and tetracycline (B) operons. The gene for fusion

protein (repressor and fluorescent protein) is expressed under the control of arabinose promoter P

BAD

The DNA of interest contains the cassette with 256 repeats of operator sequence lacO (A) or tetO (B). Binding

of repressor to the operator sequence results in focusing of fluorescence in the position of DNA of interest.

For the first time FROS was used by Robinett and co-workers to visualize chromosomal DNA

in budding  yeasts.  256  repeats  of  an  operator  sequence  from  the  lactose  operon  lacO  were
introduced to the yeast chromosome. Then the gene encoding for the LacI repressor in fusion with
GFP  was  expressed;  binding  of  the  LacI-GFP  to  the  lacO  sequence  resulted  in  the  observation  of
a fluorescent focus in the position corresponding to the subcellular localization of chromosomal DNA.
A similar system, based on tetracycline operon elements was used later by Michaelis and co-workers.
The FROS technique is now mainly applied and intensively exploited for determination of plasmid’s
localization in bacterial cells. The main advantage of this system is that it enables the observation of
molecules in living cells in different conditions and at different points of the cell cycle. Locating the
gene for a fusion protein under the control of a regulated promoter also facilitates the control over
the expression level of the gene. The only difficulty and limitation of the FROS technique is that it
requires  the  introduction  of  an  operator  sequence  into  the  sequence  of  the  DNA  molecule of
interest. The bacterial cell should also contain the gene for the fusion protein. These requirements

BAD

lacI

gfp

eyfp

tetR

lacO

tetO

lacO

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

cause the necessity of genetic manipulations before microscopic observations and are a bottleneck
of the FROS technique.

BRIDGED PROCEDURE

1. Preparation of a DNA molecule of interest (incorporation of an operator sequence) and the gene encoding

the fusion protein

2. Cell culture preparation
3. Induction of the expression of the lacI-gfp gene (time of the expression should be adjusted experimentally;

for E. coli cells the expression from the PBAD promoter usually lasts 30 min.)

4.  Inhibition of the expression of the lacI-gfp gene
5.  Additional staining of the cells (optional)
6.  Preparation of the specimen

Fluorescent in situ hybridization (FISH)

An observation of the subcellular localization of a nucleic acid can be performed not only

in living cells (with FROS) but also in fixed cells - by using FISH technique. FISH was developed in the
late 1960s and utilizes the hybridization of radiolabeled probes to a complementary sequence of
the investigated nucleic acid. Nowadays, fluorescently labeled probes are commonly used for FISH
experiments. The probe can be a restriction fragment that is labeled at one end by incorporation of
a modified nucleotide (

☼-dNTP) by the terminal deoxinucleotide transferase (TdT) (Fig. 15) or during

a 3'-end filling reaction with the use of Klenow fragment of the DNA polymerase I.

Apart from the preparation of the probes, fixation of the cells is required during a FISH

procedure  and  consists  of  incubation  with  a  crosslinking  agent  (e.g.  paraformaldehyde)  and
subsequent dehydratation of the cells in alcohol baths (70%, 90% and 96% ethanol). The cells are
prepared on a glass slide what facilitates the following microscopic observations. Before the probes
are added to the cells both probes and the investigated DNA are denatured by heating the samples
and  then  hybridization  is  continued  at  42°C  for  16-20  hours.  Next,  the  excess  of  the  probes  is
removed  by  simple  washing  of  the  glass  slide  and  then  additional  staining  of  the  cells  can  be
performed.

BRIDGED PROCEDURE

1. Preparation of the probes (fluorescent labeling with tagged nucleotide

☼-dNTP)

2.  Cell fixation (formaldehyde, paraformaldehyde)
3.  Permeabilization ( lyzozyme, detergent) (optional)
4.  Cell dehydration (ethanol baths)
5.  Denaturation of the probes and the nucleic acid within the cells
6.  Hybridization
7.  Removal of the excess of the probes
8.  Additional staining (optional)
9.  Microscopic observations

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Fig. 15. A scheme of cells (A) and fluorescent probe preparation (B) and abridged FISH procedure (C). The cells

are fixed with for e.g. paraphormaldehyde and the cell wall and membranes are permeabilized (A) The probe is

prepared by addition of fluorescently labeled nucleotide to the 3’ end of short DNA fragment, containing

sequence complement to the DNA of interest. The reaction is catalyzed by terminal deoxynucleotide

transferase (B). Then the probes are mixed with the cells (C) and the sample is heated, making possible the

denaturation of DNA, both the probe and the investigated DNA. Next the cells are incubated allowing

the hybridization of the probe with DNA of interest within the complement region. After that cells can be

additionally stained with fluorescent dyes (e.g. DAPI) and prepared for microscopic observations.

The procedure is very similar for both prokaryotic and eukaryotic cells, therefore it is

commonly used in microbiology, genetic diagnostics and tumor biology. It allows to detect even very
small alterations (deletion, duplications, rearrangements) in the genetic material, therefore it is
a popular method of karyotyping (visualization of a cell’s chromosomes). FISH can also be utilized for
comparing samples from different organisms and finding the differences in a gene arrangement.

Nucleic acid localization via its binding proteins labeling

Both FROS and FISH are methods in which the nucleic acid is tagged with a fluorescent protein

or a labeled nucleic acid fragment, respectively. The position of the DNA or the RNA within a cell can
also be determined by staining proteins that naturally bind the nucleic acid molecule. It can be
achieved by expression of a gene for a fusion protein (protein of interest and a fluorescent one) or by
immunostaining. The first method is utilized in living cells and is very similar to the FROS technique.
It also requires a fusion between the gene for the protein of interest and the fluorescent one, but in

5’-

- 3’

dCTP

5’-

3’ end labeling
by TdT

fixation and
permeabilization

denaturation

hybridization

microscopic observations

5’-

- 3’

dCTP

5’-

3’ end labeling
by TdT

fixation and
permeabilization

denaturation

hybridization

microscopic observations

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

contrast  to  the  FROS,  it  does  not  require  cloning  of  a  binding  sequence.  This  makes  the  method
easier  than  FROS,  however,  not  all  proteins  fused  to  a  fluorescent  one  can  remain  active,  like
a repressor (e.g. LacI) does. This limitation can be omitted in a immunostaining technique, but the
observations  are  performed  on  fixed  cells,  similarly as  in  the  FISH  method.  Instead  of  the  labeled
probes it requires utilization of labeled antibodies that recognize the protein of interest.
Unfortunately, both techniques stain the nucleic acid indirectly and what is observed is the protein’s
subcellular  localization.  It  can  only  be  suspected  that  the  localization  of  the  nucleic  acid  within
the cell is equal to the subcellular position of the area covered with the nucleic acid binding protein.

Confocal microscopy

Although fluorescent light microscopy has many advantages, it is very difficult to obtain images

of thick specimens that emit light. What is more, the light from above and below the focal plane can
reduce  the  contrast  what  is  observed  as  blurring  of  an  image.  These  limitations  are  omitted  in
confocal  microscopy,  which  enables  to  obtain  3D  sharp  images  of  specimens  as  thick  as
50 micrometers or more. The idea of such a microscope was created and then patented in 1957 by
Marvin Minsky. It is based on placement of the light source behind the pinhole in a such a way that it
forms a point source of light, focused on one spot of specimen. Then the reflected light is focused by
the objective lens at the image plane (Fig. 16). The presence of a non-transparent pinhole aperture
enables  only  light  reflected  from  the  specimen  to  pass  to  the  detector  and  rejection  of  the  light
coming from above and below of a focal plane. To obtain the information of a whole specimen the
spot is scanned across the image and date are collected sequentially.
Nowadays one of the most popular confocal microscopes is epi-illumination, in which light emitted
by the laser passes though the pinhole aperture, scans the specimen and passes through the second
pinhole  aperture  positioned  in  front  of  the  detector.  The  result  of  specimen  observations  with
confocal microscope is a 3D image and the specimen can be either live or fixed cells and tissues.

Fig. 16. A scheme of confocal microscope. The path of the light is

marked by black arrows. The light from the source of light passes

through the pinhole, then the light is collected by the condenser lens

and is focused as a spot within the specimen. Light emitted by the

sample passes through the objective lens and the pinhole and finally

reaches the detector.

Pinhole

Specimen

Objective

Light source

Specimen

Detector

Condensor

lens

Pinhole

Specimen

Objective

Light source

Specimen

Detector

Condensor

lens

Specimen

Objective

Light source

Specimen

Detector

Condensor

lens

Condensor

lens

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Scanning techniques

Confocal microscopes differ in beam scanning techniques and the single-beam scanning and

multiple-beam scanning microscope can be distinguished. The first one is used in most laser scanning
confocal microscopes (LSCM) and can use galvanometer mirrors, acousto-optic devices or oscillating
mirrors which cause the focusing of a single beam on the specimen, what enables to sequentially
point-scan  a  region  of  interest.  Unfortunately  these  kinds  of  microscopes  have  limited  speed  of
scanning, therefore they are unsuitable for observations of millisecond dynamic events. In multiple-
beam scanning microscopes (spinning disk microscopes) these limitations are omitted because of a
spinning Nipkow disc application. It is a disk containing an array of pinholes and microlenses (Fig. 17)
that  enables  the  usage,  in  parallel,  an  array  of  beams,  what  results  with  faster  scanning  of
the specimen.  Spinning  disk  microscopes  have  found  application  in  observations  of  high  speed
intracellular events.

Fig. 17. The improved Nipkow disk. The first disk

contains microlenses and the second one contains

pinholes. The path of the light through the disk is

shown by the black thin arrows. The direction of the

disk movement is indicated by the thick black arrow.

Detectors used in confocal microscopy

In confocal microscopes different types of detectors can also be used, which include

photomultipliers,  photodiodes  or  charge-coupled  devices  (CCDs).  All  of  them  are  highly  sensitive
photon detectors, since the amount of light available for image formation is reduced in a confocal
microscope  because  of  the  exclusion  of  light  from  fluorescent  structures  located  away  from  the
objective focal plane.

The most popular one is photomultiplier, which is a photosensitive surface that catches

the photons and then transfers the absorbed photons’ energy into a stream of photoelectrons.
The energy transition occurs in a photocathode that emits electrons. Then electrons are multiplied
on dynodes (chain of electron multipliers) and finally reach the anode (Fig. 18)

Fig. 18. Schematic representation of a photomultiplier. The photocathode, dynodes and anode are marked on

the scheme. The arrows indicate the path of the light and photoelectrons. (based on Engstrom).

photocathode

dynode

anode

light

electrons

photocathode

dynode

anode

light

electrons

microlenses disk

pinholes disk

microlenses disk

pinholes disk

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Specimen preparation

The preparation of the specimen for confocal microscopic observations is very similar as for

a conventional  wide  field  microscope.  However,  because  the  objects  observed  with  a  confocal
microscope  are  thick,  their  preparation  should  not  damage  the  3D  structure  of  the  specimen.
To avoid specimen deformations very often a spacer is used as a fishing line or a piece of coverslip is
put between the slide and the coverslip. The specimen for these kinds of observations can be either
live  or  fixed  cells  and  tissues,  depending  on  their  transparency.  The  fixation  of  the  sample  can
increase  the  penetration  of  the  specimen  by  the  laser  beam  and  reduce  opacity  and  turbidity  of
the object. If the penetration of the light is not enough to image all the sections of the specimen,
the fixed  sample  can  be  cut  using  a  microtome  and  each  section  can  be  imaged  separately.
The fixation is very often combined with permeabilization of the specimen, what facilitates the later
sample staining. The permeabilization of the cells can be achieved by incubation with DMSO, Triton
X-100, saponin or deoxycholate. Then the additional staining can be performed with fluorescent dyes
or labeled antibodies and due to the photobleaching effect also liquid embedding media like glycerol
should be used.

BRIDGED PROCEDURE

1.  Fixation
2.  Permeabilization
3.  Staining
4.  Mounting
5.  Observations

Electron microscopy (EM)

Despite the fact that the simplicity of light microscopy makes it the most popular technique to

visualize  biological objects,  it  is  connected  with some  limitations  that  disable  it  in  observations of
small macromolecules.  These  restrains  can  be overcome in electron microscopy, where instead of
light,  a  beam  of  electrons  generates  the  image  of  very  small  objects.  The  magnitude  and
the resolution obtained with EM are 10 000 000× and 0,2 nm, respectively. The source of electrons is
a  tungsten  cathode,  followed  by  an  anode  that  accelerates  the  electrons  (Fig.  19).  When
the electrons pass through the specimen, they are focused by a series of magnetic lenses. At the end
of their pathway there is a fluorescent screen or a photographic plane; the electrons interact with
these materials as photons and generate an image of the specimen.

The type of EM described above is the transmission electron microscopy (TEM), since

the beam  of  electrons  is  transmitted  through  the  specimen  and  its  image  is  generated  by  the
particles  that  pass  through  it.  The  second  most  popular  EM  technique  is  the  scanning  electron
microscopy (SEM), in which the electrons scan the specimen and a signal from every scanned spot is
collected, amplified and displayed. There can be three types of data collected: second electrons (SE),
backscatter electrons (BE) and characteristic X-rays, however, very rarely one instrument contains all
three types of detectors. The obtained image has a characteristic three-dimensional aspect resulting
from highlights and shadows of the scanned surface. It has weaker parameters of the obtained image
than the TEM (resolution 10-20 nm; magnification 500 000×), but it enables to observe thick objects
and to generate an image of their surface. The SEM is usually used for observation of whole cells and
tissues.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Fig. 19. A scheme of the electron microscope.

The elements as source of electrons, condenser,

intermediate and projector lens are marked in the

picture. The path of the electron beams is indicated by

gray arrows. The intermediate images and final image

are shown as black arrows.

Specimen preparation

The specimen preparation from different biological materials is very similar and consists of

adsorption on a support matrix (carbon-coated grid), drying and contrast addition. What is very
important for TEM, the specimen must be very thin, less than 20 nm.

Due to the fact that also the support matrix can have impact on the quality of the image,

the grid preparation is the very first step in an EM procedure. The grid is usually made of copper
coated with a support film. The most common one is a carbon film, since it forms a very thin layer
and does not influence the images. Unfortunately, carbon creates a hydrophobic environment and
before use it must be subjected to ionizing gases or chemical treatment to become hydrophilic.

Then the sample can be prepared. The first step of this procedure is the sample adsorption on

a supporting matrix. When a nucleic acid is going to be observed there are two methods of molecule
adsorption: spreading with the use of a basic protein film or a direct adsorption. In the spreading
method  usually  it’s  the  cytochrome  c  that  is  mixed  with  a  nucleic  acid.  The  positively  charged
cytochrome  c  complexes  the  negatively  charged  nucleic  acids  and  forms  a  film  at  the  water-air
interface. Then such a mixture can be spread on the carbon-coated grid. The nucleic acid can also be
directly  adsorbed  on  a  mica  support,  which  is  a  more  common  method.  It  requires  deposition  of
the amine  groups  (pentylamine)  on  a  carbon-coated  grid  and  their  spontaneous  interaction  with
the nucleic  acids.  Molecules  can  also  be  bound  to  carbon  via  mediators  such  as  the  polylysine
(polymer with positively charged groups) or divalent cations (Mg

Electron

source

Specimen

Condensor

lens

Objective

lens

Intermediate

lens

Projector

lens

Anode

First intermediate image

Intermediate image

Final image

Electron

source

Specimen

Condensor

lens

Objective

lens

Intermediate

lens

Projector

lens

Anode

First intermediate image

Intermediate image

Final image

Condenser lens

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Since contrast in the EM depends on the atomic number of the atoms in the sample, and

the biological  molecules  such  as  a  nucleic  acid  or  proteins  have  low  value  of  this  parameter,  it  is
required  to  stain  them  with  heavy  metal  salts  (salts  of  osmium,  uranium,  lead)  possessing  a  high
atomic number. When the beam of electrons meets the heavy metal atoms it is diffracted under such
a large angle that it cannot enter the imaging lanes. The staining can be positive, when the heavy
metal ions interact with the molecule what results in contrasting of the molecule, or can be negative,
when  the  macromolecule  is  usually  unstained  in  contrast  to  the  stained  surroundings.  The  most
popular  method  of  nucleic  acid  staining  utilizes  uranyl  acetate,  which  forms  simple  salts  with  the
phosphate groups of DNA. This compound can also be used for staining proteins (it is then performed
in  lower  pH  than  the  one used  for  DNA)  and  RNA.  For  enhancing the obtained  signal  to  generate
a higher  quality  image, antibodies  can  also  be  used.  They can  bind  to a  protein,  simply  increasing
the size  of  the  investigated  object  or  they  can  be  conjugated  with  gold  particles,  what  improves
the detection (Immunogold Electron Microscopy).
Apart  from  staining  the  contrast  can  be  enhanced  by  a  metal  shadowing  technique.  It  consists  of
spraying particles of a vaporized metal onto the whole specimen. This technique is commonly used
to  emphasize  the  details  of  biological  structures.  Platinum  is  most  commonly  used  in  the  metal
shadowing; first it is melted and then evaporated from a heated electrode.

Cryo-electron microscopy (CEM)

The basic limitation of EM is that it requires preparation of a dry specimen. It makes impossible

the  observation  of  molecules  within  a  buffer.  This  restriction  can  be  avoided  in  a  cryo-electron
microscopy. In CEM the specimen is observed at liquid nitrogen or liquid helium temperatures, what
prevents evaporation of water. The specimens are usually prepared by vitreous-ice technique, during
which  a  sample  placed  on  a  grid  is  rapidly  frozen.  The  specimen  can  also  be  frozen,  cut  with
a diamond  knife  into  thin  pieces  (40-200  nm)  and  then  placed  on  the  grid.  The  low  temperature
during  imaging  ensures  additional  protection  of  the  specimen  from  radiation  damage.  Results
obtained  with  the  CEM  can  be  reconstructed  into  three-dimensional  images  of  the  analyzed
molecule.  Compared  to  the  negative  staining  and  the  metal  shadowing  of  the  specimen  in  EM,
the CEM image is a superimposition of all of the densities in the sample what results in more actual
data.

Atomic force microscopy (AFM)

Although electron microscopy is characterized by resolution as high as 0,2 nm sometimes it is

insufficient  to  observe  very  small  objects.  The  atomic  force  microscopy  enables  to  observe  even
the arrangement of individual atoms in a sample with a routine resolution higher than 0,1nm. It was
developed  in  1986  on  the  basis  of  the  scanning  tunneling  microscope.  In  AFM  the  image  is  not
generated by focusing light or electrons on a specimen but by interaction between the surface and a
sharp probe (tip) of a cantilever. It is very often said that the probe “feels” the sample in AFM. During
the sample scanning, a map of its surface is generated and then the collected data is transformed
into  an  image.  There  are  three  basic  elements  of  an  AFM  instrument:  a  piezoelectric  transducer,
a force sensor and a feedback control. The first is responsible for the movement of the tip over the
scanned  surface.  The  piezoelectric  properties  of  this  element  result  in  changes  in  its  geometry
without changes in the volume under the influence of the applied electrical potential (Fig. 20).

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Fig. 20. The shape of piezoelectric disk depending on

applied voltage. Piezoelectric material causes that the

disk changes its shape with the constant volume

retention when the voltage potential is applied. (based

on Eaton and West, modified).

The piezoelectric transducer, also called a piezoelectric scanner is usually made of ceramic

materials such as amorphous lead barium titanate (PdBaTiO

) or lead zirconate titanate

(Pd[Zr

1-x

). It changes the electric potential into a mechanical motion and depending on its shape

it can have more or less motion. Usually it has the shape of a tube (it is present in over 75% of all
AFMs in use).

The second important element of the AFM is the force sensor, which measures the force

between the tip and the surface of the specimen. It can measure forces as low as 10 pN. Most of
the AFM microscopes contain an optical lever force sensor, which consists of a laser focusing light on
the cantilever, that reflects this light to the detector (Fig. 21).

Fig. 21. Scheme of optical lever sensor in the Atomic Force

Microscope. Cantilever is the end with a tip that scans

the surface of the specimen. The up-and-down movements

of the cantilever change the distance between the

cantilever and the detector, what influences the place on

the detector when reflected light falls. (based on Eaton and

West, modified).

Then the signal is directed to the feedback control, which controls the force between the probe and
the  specimen’s  surface  and  the  movement  of  the  probe.  It  moves  the  probe  in  such  way  that
the force is constant. For example, if the force sensor detects an increase in the force, it means that
the probe is in closer contact with the surface and it should be moved upwards.
The feedback control enables keeping a proper distance between the sample and the tip. Depending
on  the  mode  type,  the  sample’s  surface  can  be  in  direct  contact  with  the  tip  (contact  mode),
the cantilever with the tip can oscillate over the surface and contact it only at the very end of every
downward movement (tapping mode) or does not contact it at all (non-contact mode).

The specimen preparation

The AFM technique enables to observe different objects. The samples that are to be prepared

can  be  dry  (air-AFM)  or  immersed  in  a  liquid  solution  (liquid-AFM),  what  gives  the  possibility  to
observe biological structures in their natural environmental conditions. What is very important the
sample  must  be  rigidly  adhered  to  a  smooth  substrate  since  movement  of  the  sample  during  the
scanning can hinder the generation of a high resolution image. The smaller molecules the smoother
substrate must be used. The most popular substrates are glass, mica or silicone oxide and sometimes
they are used with additional chemicals (e.g. poly-L-lisine, polyethyleneimide, divalent cations) that
facilitate the sample-substrate interaction. Mica is the most often used in biological applications and
like  in  the  EM  specimen’s  preparation,  it  is  often  coated  with  carbon,  what  yields  hydrophobic
surface suitable for immobilization of hydrophobic molecules e.g. DNA. For DNA also the addition of

detector

cantilever

las

ght

detector

cantilever

las

ght

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

divalent cations, such as Mg

, Mn

, Zn

, Ni

, can improve the DNA binding to the mica.

Interestingly, the monovalent cations (

, Na

, Li

) can inhibit the proteins-mica binding.

Sometimes

a special gold surface is prepared as it is inert against oxygen and free radicals species and can be
easily modified to facilitate the molecules’ adhesion. Molecules can also be bound covalently to
an appropriately prepared substrate. Sometimes special linkers are used that mediate between
the molecule and the substrate ensuring the mobility of the molecule, which can freely interact with
complementary molecules.

There are different methods for placing the sample on the substrate. For the air-AFM, a drop

of molecule solution can be deposited on the substrate and left to evaporate or the substrate can be
immersed in the solution and then air-dried. The sample can also be sprayed onto a substrate.
The molecules can also be directly imaged in liquid conditions without air-drying.

Applications

The AFM is a technique that gives the possibility to observe all kinds of molecules in different

conditions with an atomic resolution. It was successfully applied for imaging of biological molecules
as small as DNA, proteins or nucleoprotein complexes as well as tissues, cells and viruses. The big
advantage of this microscopic technique is that it enables reconstruction of the biological processes
and their observation, like for example the movement of RNA polymerase along the DNA template.
Moreover, it is still developed to obtain better resolution, especially with soft samples in as short
time as possible.

ITERATURE

Andreeff M, Pinkel D, 1999, Introduction to fluorescence in situ hybridization: principles and clinical

applications, Wiley-Liss.

Bozzola J.J. and Russell L.D, 1999, Electron microscopy. second edition, World Headquarters.

Braga P.C. and Ricci D, 2004, Atomic force microscopy: Biomedical methods and Aplications. Human Press

Claxtone N. S, Fellers T. J, Davidson M. W, 2005, Laser Scanning Confocal Microscopy,

http://www.olympusfluoview.com/theory/LSCMIntro.pdf

Conn P.M, 2010, Techniques in confocal microscopy, Academic Press.
Dykstra M.J. and Reuss L.E, 2003, Biological Electron Microscopy: theory, techniques and troubleshooting.

second edition.

Eaton P. and West P, 2010, Atomic force microscopy, Oxford University Press.
Engstrom R.W, 1980, Photomultipiler handbook. RCA Corporation.
Fuller S, 1999, Cryo-electron microscopy:taking back the knight. Microbiology Today 26: 56-58.

Kirat K.E, Burton I, Dupres V, Dufrene Y.E, 2005, Sample preparation procedures for biological atomic force

microscopy, Journal of Microscopy 218(3): 199-207.

Liehr T, 2009, Fluorescent in situ Hybridization (FISH) – Aplication Guide, Springer.
Michaelis C, Ciosk R, Nasmyth K. 1997 Cohesins: chromosomal proteins that prevent premature separation of

sister chromatids. Cell. 91(1): 35-45.

Muller M, 2006, Introduction to confocal fluorescence microscopy. Second edition, SPIE.
Pogliano J, Ho TQ, Zhong Z, Helinski DR. 2001 Multicopy plasmids are clustered and localized in Escherichia coli.

Proc Natl Acad Sci USA. 98(8): 4486-91.

Price R.L. and Gerome W.G, 2011, Basic Confocal Microscopy, Springer.
Verheust C, Helinski DR. 2007 The incC korB region of RK2 repositions a mini-RK2 replicon in Escherichia coli.

Plasmid 58(2): 195-204.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 8. Two dimensional electrophoresis of nucleic acids

Classical nucleic acid electrophoresis enables to separate fragments which differ in molecular

mass. It is based on the ability of the negatively charged molecules to migrate in the electric field to
the anode. Since the migration of nucleic acid is performed in porous agarose or polyacrylamide gel,
the  smaller  fragments  migrate  easier  through  the  pores  and  can  be  separated  from  fragments  of
higher molecular mass that migrate slower. Electrophoresis is a basis of different methods used in
restriction mapping, molecular diagnostics or genetic fingerprinting (e.g. RFLP-PCR; see Chapter 1).
However, the resolution of one dimensional electrophoresis is limited. Therefore the separation of
samples  in  the  second  dimension  can  evidently  improve  the  method  and  quality  of  the  obtained
results.  Two-dimensional  electrophoresis  (2-DGE)  was  discovered  over  fifty  years  ago  to  separate
peptides  and  proteins.  Then  it  was  also  applied  for  separation  of  t-RNAs,  large  fragments  of  high
molecular weight RNA, DNA restriction fragments or DNA replication intermediates. In this technique
two  electrophoretic  separations  of  molecule  mixtures  are  performed.  The  first  separation  is
performed  in  agarose  or  polyacrylamide  gel  and  then  further  electrophoresis  is  conducted  but  in
perpendicular  direction  to  the  first  one.  In  case  of  nucleic  acids  the  first  dimension  separates
according  to  the  molecular  mass  and  the  second  one  according  to  the  structure.  In  2-DGE,  DNA
structures such as Y-shaped, X-shaped or circular molecules can be easily distinguished.

2-DEG of replication intermediates

One of the most common application of two-dimensional DNA gel electrophoresis concerns

separation of replication intermediates. It requires the digestion of DNA molecules and separation of
the  obtained  fragments  in  two  dimensions.  Very  often  the  sample  preparation  comprises  also
an additional step - the BND-cellulose chromatography of DNA fragments. It provides the enrichment
of  the  sample  in  DNA  fragments  containing  replication  bubbles  since  BND-cellulose  binds  single
stranded DNA.

The first electrophoresis is performed in neutral conditions and separates DNA fragments

according  to  mass.  It  is  performed  in  1×TBE  buffer  in  a  low  agarose  concentration  gel  (0,4%)  to
minimize dependence of mobility on structure. The first dimensional gel is run at low voltage (1V/cm)
for  about  15h,  however  for  larger  DNA  fragments  (>6kb)  it  is  recommended  to  even  reduce
the voltage since branched intermediates bigger in size are more likely to have retarded mobility.
The  second  dimension  electrophoresis,  resolving  the  DNA  fragments  according  to  shape,  can  be
performed  in  neutral  or  denaturing  conditions.  A  neutral  electrophoresis  is  prepared  when
the replication origin region is going to be identified. A gel for electrophoresis in second dimension
has  higher  agarose  concentration  (1-1,3%)  and  addition  of  ethidium  bromide  (0,5µg/ml).  It  is  run
at higher voltage (5-9V/cm) in 1×TBE buffer supplemented with ethidium bromide (0,5µg/ml). Based
on the shape of the obtained separation curve, it can be estimated what the DNA fragment looks like
and if it contains a replication origin region (Fig. 21). The presence of characteristic arcs created by
migrating bubble- or Y-shaped DNA fragments can confirm the occurrence of replication forks.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Fig. 21. Scheme of replication intermediates separated in gel during neutral 2D electrophoresis. In the first

dimension intermediates are separated based on their size and then the separation in the second dimension is

based on size and shape. The pattern of their separation, depending on shape of intermediates: simple Y (A),

bubble (B), double Y (C), bubble changing into Y (D) is shown on the scheme. (based on Brewer and Fangman,

modified).

If the direction of replication forks movement is going to be determined, a second dimension

gel should be performed in denaturing conditions. Alkaline buffer (AEB buffer containing 30-40mM
NaOH  and  2mM  EDTA)  secures  such  an  environment.  It  should  be  remembered,  however,  that
agarose cannot gel under such conditions, therefore it should be prepared in water and then soaked
in  the  AEB  buffer.  The  gel  for  the  second  dimension  in  denaturing  conditions  has  higher  agarose
concentration,  but  the  voltage  during  electrophoresis  should  be  reduced  in  comparison  to  the
neutral one. This is due to the higher conductivity of AEB than TBE buffer, what can cause heating
and  distortion  of  the  gel.  Also  ethidium  bromide  is  omitted  since  it  does  not  work  in  high  pH
conditions.  Therefore  to  visualize  the  obtained  results  southern  blotting  is  usually  performed  and
DNA fragments, after transfer to a nylon membrane are hybridized with radiolabeled probes. As in
neutral/neutral  2-D  electrophoresis,  the  shape  of  the  curve,  formed  by  separating  nucleic  acid
fragments, brings the information about the replication intermediate features. Denaturing conditions
cause four single stranded DNA fragments to arise from every fragment containing replication forks.
The migrating parental strands form the horizontal line and nascent strands form an arc. Depending
on the length of nascent strands the arc has a characteristic shape (Fig. 22).

Fig. 22. Structure of replicating molecules and the

shape of the nascent strand arc. The restriction

fragments containing the origin region in different

positions or the intact circular molecule separating

in 2D polyacrylamide gel in neutral/denaturing

conditions (based on Nawotka and Huberman,

modified).

Since these two techniques, neutral/neutral and neutral/alkaline two-dimensional

electrophoresis, deliver information that complement one another it is recommended to perform
both experiments. Such a strategy can result in a complete picture of the replicating molecule.

SIMPLE Y

BUBBLE

DOUBLE Y

BUBBLE

 Y

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

FIRST

SIMPLE Y

BUBBLE

DOUBLE Y

BUBBLE

 Y

SIMPLE Y

BUBBLE

DOUBLE Y

BUBBLE

 Y

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

1kb

2kb

FIRST

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Two-dimensional Gene Expression Fingerprinting (2-D GEF)

There are a lot of different methods applied to gene expression analysis. One of them is gene

expression  fingerprinting  (GEF),  in  which  non-overlapping  sets  of  3’-terminal  cDNA  restriction
fragments are separated on a polyacrylamide gel. The further digestion of the obtained separation
fragments  and  electrophoresis  in  the  second  dimension  improves  the  resolution  of  the  initial
method. It can be used in preliminary gene profiling of a genome project or in searching for novel
expressed sequences.

BRIDGED PROCEDURE OF

2-D

GEF

1.  RNA isolation
2.  cDNA synthesis with biotinylated primer
3.  Synthesis of second strand of cDNA by E. coli polymerase I
4.  Primary restriction digestion
5.  Isolation of DNA fragments via biotin and adaptor ligation (contains a sequence recognized by the primer)
6.  PCR reaction
7.  First dimensional polyacrylamide gel electrophoresis
8.  Elution of separated DNA fragments and their amplification
9.  Second restriction digestion
10. Second dimensional electrophoresis and data analysis

The major difficulty that makes this method time- and labor-consuming is restriction digestion

of every isolated DNA fragment (see point 7 and 8 of the procedure) and its further separation and
analysis. Fortunately, the progressive automation of experimental procedures increase the possibility
of improving this method in the future and make it easier to perform.

Two-dimensional gel shift assay

The canonical gel shift assay (see Chapter 3) allows to analyze the nucleic acid fragment in

context  of  its  binding  by  the  protein.  It  is  a  simple  technique  where  a  particular,  small  region  is
suspected  to  be  bound  and  going  to  be  analyzed.  Unfortunately,  if  the  precise  site  recognized  by
the protein is unknown and is localized within the large nucleic acid fragment it can be difficult to
identify  the  protein  binding  site  by  GMSA.  In  such  a  situation,  the  division  of  large  fragment  into
smaller ones and verification each of them separately would be required. These kinds of limitations
can be omitted when two-dimensional gel electrophoresis is utilized. It can be performed with, for
instance entire plasmid DNA, that should be digested to obtain short fragments and labeled in the
next step by incorporation of modified nucleotides (

P-dNTP; Cy3-dNTP). Then the mixture of nucleic

acid  fragments  is  incubated  with  the  protein  of  interest  and  separated  on  a  polyacrylamide  gel
(6-7,5%) in 1×TBE buffer. Next the gel should be heated (75°C) to allow disruption of nucleoprotein
complexes  and  a  high  temperature  (60°C)  should be  kept  during  the  entire  electrophoresis  in  the
second  dimension.  2-DGE  is  performed  in  the  same  gel  but  rotated  90°.  After  electrophoresis,
visualization  of  the  results  should  be  performed.  If  any  restriction  fragments  are  bound  by
the protein, they run ahead of the diagonal in the second dimension (Fig. 23). Therefore it is easy to
indicate  the  region  of  DNA  molecule  forming  a  nucleoprotein  complex,  isolate  it  and  subject  to
further investigation.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Fig. 23. Two-dimensional electrophoresis of gel shift assay. (A) The scheme of the gel performed in first

dimension. The arrow indicates the shift of the band representing the nucleoprotein complex. (B) Scheme of

the same gel resolved in the second dimension in higher temperature allowing a disruption of the complex. The

DNA fragment released from the complex migrates ahead of the diagonal in the second dimension. (based on

Boffini and Prentki, modified).

ROUBLESHOOTING

1. If the 2D pattern is distorted

− fresh, degassed reagents for gel preparation should be used
− prolong the gel polymerization time

2. If horizontal streaking or incompletely focused spots are observed:

− check the salt concentration and reduce it when required
− check the amount of loaded sample and reduce it when required

3. If no bands are visible after electrophoresis :

−  the amount of used nucleic acid should be verified and adjusted to detection level
−  the labeling reaction should be controlled and probably repeated
−  the nuclease inhibitors could be added to prevent nucleic acid degradation

ITERATURE

Boffini A. and Prentki P, 1991, Identification of protein binding sites in genomic DNA by two-dimensional gel

electrophoresis, Nucleic Acids Research 19(7): 1369-1374.

Brewer B.J. and Fangman W.L, 1987, The Localization of Replication Origins on ARS Plasmids in S. cerevisiae,

Cell 51: 463-471.

Cobuzzi R. J, Burhans W. C, Berman T.A, 1996, Inhibition of Initiation of Simian Virus 40 DNA Replication in

Infected BSC-1 Cells by the DNA Alkylating Drug Adozelesin. J Biol Chem 271(33): 19852-19859.

Nawotka K.A.and Huberman J.A, 1988, Two-Dimensional Gel Electrophoretic Method for Mapping DNA

Replicons, Mol Cell Biol 8(4): 1408-1413.

Shmelkov S.V, Visser J.W.M, Belyavsky A.V, 2001, Two-Dimensional Gene Expression Fingerprinting, Analytical

Biochemistry 290, 26-35.

Stein M. and Varricchio F, 1974, Separation of t-RNAs by two-dimensional polyacrylamide gel electrophoresis,

Analytical Biochemistry 61(1): 112-119.

Vertical gel

Horizontal gel

DNA

protein

DNA

protein

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 9. Methods for origin region determination

The replication process starts in a specific region called origin. It contains sites for initiator

protein binding and an AT-rich region, where the local melting of dsDNA occurs. In different DNA
molecules there can be one (Prokaryotes, Archea), two (Archea) or multiple (Archea, Eukaryotes)
origins indicated. Techniques used for the origin determination can be divided into in silico, in vivo
and in vitro methods.

In silico methods

The relative nucleotides skews

The simplest in silico method used to determine the position of a replication origin in

the bacterial chromosomes takes advantage of the asymmetry in nucleotide composition between
the leading and lagging strands of the DNA. This differences in the GC and the AT content are usually
presented as relative nucleotide skews (e.g. CG skew = (C-G)/(C+G)) and result from different
mutation rates connected with the architectural asymmetry at the replication forks and the higher
accuracy of the leading strand replication. The relative skew value is a normalized difference in the
content of the complementary nucleotides, which changes the sign crossing of a replication origin.
It can be illustrated graphically with the chromosome walking technique, in which consecutive
nucleotides are analyzed and depending on their character the curve is drawn. For example, G results
in a movement about one unit to the top on the graph, whereas C results in a movement down and A
or T - one unit forward. Such a procedure results in obtaining a diagram of differences between G
and C content (Fig. 24).

The relative nucleotide skews is used in many software tools designed for origin prediction, for

example in the Oriloc, CG-software, Z-curve and Ori-Finder programs. However, the analysis of
chromosome asymmetry gives only the approximate origin position within the analyzed genome’s
sequence, simply indicating the region with higher than average AT content.

Fig. 24. A graphic analysis of chromosome

asymmetry reveled with the chromosome

walking method. A curve shows the

dependence between the nucleotide

position within the chromosome and an

asymmetry (the difference in content of G

and C residues). The extremes on the curve

correspond to origin and termination

regions in the chromosome and sites

where leading strand change into lagging

one.

ter

position in the chromosome
(nucleotide)

(

-C)

leading

strand

leading

strand

lagging

strand

ori

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Localization of genes for the replication initiation protein and its binding sites

The determination of genes for initiator proteins can be used for origin region prediction.

It was  shown  that  the  gene  for  bacterial  replication  initiator  protein  DnaA  is  found  in  almost  all
bacterial chromosomes and the origin for replication is usually located upstream or downstream of
this  gene.  The  same  situation  was  observed  for  Archea,  which  possesses  genes  for  replication
initiation proteins Orc1/Cdc6 close to the origin region. Unfortunately this method does not applied
to eukaryotes, while the origins for DNA replication are found throughout the genomes (for example
in  yeasts  there  are  over  500  origins  in  a  genome)  and  the  genes  for  initiator  proteins  cannot  be
present in proximity of all of them.

The more universal feature for origin region prediction can be the presence of replication

initiation protein binding sites. The region where the initiator form a nucleoprotein complex usually
is contained within the origin of both prokaryotes and eukaryotes. The binding sites for bacterial
initiator DnaA, archeal Cdc3 and MCM proteins and origin recognition complex (ORC) in eukaryotes,
were all found near the site where the replication bubble is formed and replication starts.

In vivo methods

Construction of minichromosomes

This is the most popular method for in vivo origin region determination. It is based on

the construction  of  minichromosomes  -  vectors  containing  particular  regions  of  a  chromosome.
The chromosome is digested with restriction enzymes to obtain DNA fragments that can be cloned
into the vector, which does not contain any origin region. If the inserted chromosome fragment can
support  replication  of  the  plasmid  in  bacterial  cells  this  could  indicate  that  the  insertion  contains
a replication origin. Unfortunately, this method is time consuming since a genomic library needs to
be  constructed  and  then  it  should  be  tested  in  the  bacterial  cells.  It  also  only  narrows  down  the
region where the origin can be found but does not indicate precisely the origin site.

Chromatin immunoprecipitation assay (ChIP)

Another method that can help to determine an origin region in vivo, is the chromatin

immunoprecipitation assay. Via the immunoprecipitation of replication proteins, that bind specific
sequences within an origin, the whole region can be estimated. For details see Chapter 6.

In vitro methods

The in vitro confirmation of the site where the replication starts can be achieved with such

techniques as:

−  two-dimensional gel electrophoresis (for details, see Chapter 8)
−  electron microscopy (for details, see Chapter 7)
−  GMSA assay (for details, see Chapter 3)
−  footprinting analysis (for details, see Chapter 4)
−  Surface Plasmon Resonance (for details, see Chapter 5)

These methods allow to observe replication intermediates, determine the specific motifs

within an origin where replication proteins bind or even calculate the stoichiometry and kinetics of
a nucleoprotein complex formation.

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Frank A. C, Lobry J. R, 2000, Oriloc: prediction of replication boundaries in unannotated bacterial

chromosomes. Bioinformatics 16: 560-561.

ITERATURE

Freeman J.M, Plasterer T.N, Smith T.F, Mohr S.C, 1998, Patterns of genome organization in bacteria. Science

279: 1827.

Grigoriev A, 1998, Analyzing genomes with cumulative skew diagrams. Nucleic Acids Res 26: 2286-2290.
Lobry J.R, 1996a, Asymmetric substitution patterns in the two DNA strands of bacteria. Mol Biol Evol 13: 660-

665.

Lobry J.R, 1996b, A simple vectorial representation of DNA sequences for the detection of replication origins in

bacteria. Biochimie 78: 323-326.

Mackiewicz P, Zakrzewska-Czerwinska J, Zawilak A, Dudek M.R, Cebrat S, 2004, Where does bacterial

replication start? Rules for predicting the oriC region. Nucleic Acids Res 32: 3781-3791.

Meijer M, Beck E, Hansen F.G, Bergmans H.E, Messer W, von Meyenburg K, Schaller H, 1979 Nucleotide

sequence of the origin of replication of the Escherichia coli K-12 chromosome. Proc Natl Acad Sci USA 76:

580-584.

Messer W, 2002, The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA

replication. FEMS Microbiol Rev 26: 355-374.

Messer W, Meijer M, Bergmans H.E, Hansen F.G, von.Meyenburg K, Beck E, Schaller H, 1979, Origin of

replication, oriC, of the Escherichia coli K12 chromosome: nucleotide sequence. Cold Spring Harb Symp

Quant Biol 43(Pt 1): 139-145.

Mrazek J, Karlin S, 1998, Strand compositional asymmetry in bacterial and large viral genomes. Proc Natl Acad

Sci USA 95: 3720-3725.

Oka A, Sugimoto K, Takanami M, Hirota Y, 1980, Replication origin of the Escherichia coli K-12 chromosome:

the size and structure of the minimum DNA segment carrying the information for autonomous replication.

Mol Gen Genet 178: 9-20.

Roten C.A, Gamba P, Barblan J.L, Karamata D, 2002, Comparative Genometrics (CG): a database dedicated to

biometric comparisons of whole genomes. Nucleic Acids Res 30: 142-144.

Salzberg S.L, Salzberg A.J, Kerlavage A.R, Tomb J.F, 1998, Skewed oligomers and origins of replication. Gene

217: 57-67.

Tillier E.R, Collins R.A, 2000, The contributions of replication orientation, gene direction, and signal sequences

to base-composition asymmetries in bacterial genomes. J Mol Evol

Zhang R, Zhang C.T, 2005, Identification of replication origins in archaeal genomes based on the Z-curve

method. Archaea 1: 335-346

50: 249-257.

UESTIONS

1.  How can in vitro methods help determine origin region?
2.  How can relative nucleotide skews be illustrated graphically?
3.  The presence of which elements of bacterial chromosome indicate the origin region?

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

CHAPTER 10. Methods for in vitro analysis of replication initiation

Replication is one of the most important processes within the living cell. It ensures

the duplication  of  genetic  material  that  is  propagated  to  the  daughter  cells.  Its  initiation  requires
the subsequent formation of nucleoprotein complexes within the origin region of a DNA molecule.
That  results  in  the  melting  of  a  double-stranded  DNA  (dsDNA)  in  an  AT-rich  region  (“opening”),
unwinding of the dsDNA by a helicase and subsequent loading of polymerase subunits synthesizing
new DNA strands.

Opening

The very first effect of the initiator proteins’ binding to the sequences within the origin is

the melting  of  dsDNA  and  formation  of  a  replication  bubble.  This  process  is  commonly  named
“opening”. It depends on replication initiation proteins (Rep), that via DNA binding cause changes of
the DNA conformation leading to occurrence of ssDNA in an AT-rich region. It is also dependent on
the  sequence  of  the  AT-rich  region  and  on  its  location  within  the  origin.  Even  small  changes  in
the organization of the origin (the insertion of 6 bp between the AT-rich region and the sequences of
iterons completely inactivated the origin of the RK2 plasmid) or point mutation in the AT-rich region
result  in  opening  inhibition.  Formation  of  the  replication  bubble  can  be  directly  observed  with
electron  microscopy  (see  Chapter 7)  or  can  be  verified  in vitro,  by  the  potassium  permanganate
probing assay.

Potassium permanganate probing assay

The potassium permanganate assay is a type of footprinting method (see Chapter 4). However,

compared with the classic one, where nucleases are used, an oxidizing agent (KMnO

) is needed in

this technique. It modifies thymine residues in the single-stranded DNA and its concentration is
adjusted to obtain just one modification per molecule. This reagent does not modify the residues in
a double-stranded nucleic acid, so its action is limited to the replication bubble. Probing with KMnO

is followed by primer extension reaction with the use of a labeled primer (see Chapter 4). During this
reaction,  the  polymerase stalls  at  the modified thymine  residues on  the  template,  what  results  in
a range  of  products  of  different  length.  Based  on  the  length  of  the  products  it  can  be  estimated
where the local melting of the double-stranded molecule and formation of a single-stranded region
occurs.

In this experiment the DNA template is usually a coiled-coil form of a plasmid DNA, containing

the origin. Its incubation with initiator proteins enables open complex formation. Then the probing
reaction is performed.

BRIDGED PROCEDURE

1. Incubation of a DNA template with initiator proteins.
2. KMnO

probing.

3.  DNA purification.
4.  Labeled primer extension reaction.
5.  Electrophoresis.
6.  Visualization of the results (depends on the type of labeling).

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

Helicase assay (FI*)

When the replication bubble is formed, the next step is the recruitment of a helicase (DnaB in

E. coli) by the initiator proteins. Helicase is loaded onto the open complex and next it unwinds the
dsDNA in the 5’ to 3’-end direction. The in vitro helicase assay is performed with a coiled-coil plasmid
DNA, in the presence of the helicase and the initiator proteins. The unwinding reaction is assisted
with  other  E. coli  proteins:  HU,  gyrase,  helicase  loader  DnaC  and  SSB.  The  helicase  activity  also
requires  the  presence  of  the  ATP  and  an  ATP  regeneration  system  (creatine  kinase  and  creatine
phosphate).  The  expansion  of  the  ssDNA  region  changes  the  electrophoretic  migration  of  a  DNA
molecule  in  an  agarose  gel.  Such  a  molecule,  marked  “FI*”,  migrates  faster  when  compared  with
a coiled-coil  form  of  the  plasmid  DNA  (“FI”)  (Fig.  25).  The  presence  of  the  FI*  band  indicates the
activity of all required proteins and especially the helicase.

BRIDGED PROCEDURE

1. Incubation of the DNA template with proteins: initiator proteins (DnaA and optionally plasmid initiator),

helicase (DnaB), helicase loader (DnaC), gyrase, HU, SSB.

2. Inhibition of the helicase reaction (incubation at 65°C with EDTA and SDS solutions).
3. Agarose electrophoresis for 20h, 25 V (1% gel in a TBE buffer; it is important to not add EtBr to the gel since

it can disturb the migration pattern).

Fig. 25. Helicase assay (FI*). The scheme presents the result

of the helicase assay, in which the reaction mixture

containing a constant amount of plasmid DNA, initiator

protein and additional proteins (HU, SSB, gyrase) is titrated

with helicase. The more helicase is added to the reaction

mixture the more FI* form of plasmid DNA is visible after the

electrophoresis in the agarose gel.

In vitro replication (FII)

The replication process is conducted by a polymerase and accessory proteins. It starts within

the origin, where the replication bubble has been formed. First, a primase is loaded onto the DNA
since the DNA polymerase cannot start the synthesis without a free –OH group at the 3’-end. Then
the clamp-loading complex (ɣ complex) loads the sliding clamp (β clamp) onto the primer-template
strand. The final step is the loading of a polymerase core, which is a heterotrimer containing
the α (DNA polymerase), ε (proofreading exonuclease) and θ (stabilizes ε) subunits. The formation of
the polymerase complex is ATP-dependent, since this compound increases the affinity of the clamp-
loader for β subunits and for DNA. The hydrolysis of the ATP is triggered when ɣ complex-β clamp-
ATP binds the DNA. Then the DNA is released by the ɣ complex.

The in vitro replication assay can be performed in a crude cell extract, containing all necessary

proteins and compounds or in a reconstructed system utilizing all purified proteins. Both methods
are based on counting of radioactivity of nucleotides labeled with

H, incorporated during the DNA

synthesis. The experiment is performed very often on a plasmid template containing the region of
an origin of interest. The DNA template that is used in in vitro replication assays can also be sperm
nuclei and ssDNA replicon. The reaction mixture contains the template added to the crude cell

FIII

FII

FI*

─

helicase

Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

initiator protein (ng)

syn

thesi

)

extract  or  to  the  mixture  of  purified  polymerase  subunits,  initiator  protein,  primase  and  other
required  components.  The  crude  cell  extracts  can  be  prepared  from  bacterial  or  eukaryotic  cells
(e.g. Sacharomyces cerevisae,  Xenopus  oocytes,  HeLa).  After  the  incubation,  during  which
the replication initiation and the DNA synthesis occur, the reaction is stopped and the radioactivity is
counted  in  a  scintillation  counter.  The  results  are  referred  to  the  base  readout  where  no  DNA
template or no initiator protein was added. The obtained values are usually presented as a diagram
(Fig. 26).

Fig. 26. Dependence of in vitro plasmid replication on initiator

protein. The diagram shows the kinetics of in vitro incorporation

of [H

]dTTP depending on the amount of initiator protein added

to the reaction mixture. The replication reaction was performed

in a cellular extract containing all necessary proteins except the

initiator protein. (A) The reaction mixtures contained equal

amounts of cellular extract, DNA template, and increasing

amounts of initiator protein. (B) Control reaction without DNA

template.

BRIDGED PROCEDURE

1. Incubation of the DNA template with purified proteins/crude cell extract.

2. Stopping the reaction with a 100 mM pyrophosphate (PiroP) and a 10% trichloroacetic acid (TCA)

solution.

3. Counting the radioactivity in a scintillation counter.

ITERATURE

Abhyankar M.M, Zzaman S, Bastia D, 2003, Reconstitution of R6K DNA Replication in Vitro Using 22 Purified

Proteins, J Biol Chem 278(46): 45476-84

Doran K.S, Konieczny I, Helinski D.R, 1998. Replication origin of the broad host range plasmid RK2. Positioning

of various motifs is critical for initiation of replication. J Biol Chem 273: 8447-8453.

Kaittell B. L, Helinski D. R, 1991, Iteron inhibition of plasmid RK2 replication in vitro: evidence for intermolecular

coupling of replication origins as a mechanism for RK2 replication control. Proc Natl Acad Sci USA, 88(4):

1389-93.

Konieczny I, Helinski D.R, 1997, Helicase delivery and activation by DnaA and TrfA proteins during the initiation

of replication of the broad host range plasmid RK2. J Biol Chem 272(52): 33312-33318.

Mukhopadhyay G, Carr K.M, Kaguni J.M, Chattoraj D.K, 1993 Open-complex formation by the host initiator,

DnaA, at the origin of P1 plasmid replication. EMBO J 12(12): 4547-4554.

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Molecular biology of nucleic acid - experimental methodology

by Katarzyna Węgrzyn & Igor Konieczny

UESTIONS

1. The figure shows the result of the potassium permanganate assay. How would you interpret the obtained

results?

2. How would you verify the replication initiator protein activity?
3. What are the components of the in vitro replication reaction?

KMnO

Initiator protein

+ +

KMnO

Initiator protein

+ +

KMnO

Initiator protein

+ +

Document Outline