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WHAT IS DNA SEQUENCING ?

DNA sequencing usually involves enzymatic DNA

synthesis in the presence of base-specific
dideoxynucleotide chain terminators

dideoxynucleotide chain terminators

Determining the DNA sequence is therefore useful in

basic research studying fundamental biological
processes, as well as in applied fields such as
diagnostic or forensic research

MAXAM & GILBERT DNA SEQUENCING

(CHEMICAL DEGRADATION)

In the late 1970s, A. M. Maxam and W.Gilbert

devised the first method for sequencing DNA
fragments containing up to ≈500 nucleotides

the sequence of a double-stranded DNA molecule is

determined by treatment with chemicals that cut the
molecule at specific nucleotide positions

molecule at specific nucleotide positions

it is the early method involving base-specific chemical

modification and subsequent cleavage of DNA

most of the chemicals used in chemical degradation

method are toxic and hazardous to the health of the
researchers doing the DNA sequencing

CHEMICAL DEGRADATION METHOD

PROCEDURE

four samples of an end-labeled DNA restriction fragment are
chemically cleaved at different specific nucleotides

the resulting sub-fragments are separated by agarose

gel electrophoresis and the labeled fragments are

detected by autoradiograph.

the sequence of the original end-labeled restriction fragment

can be determined directly from parallel electrophoretograms
of the four samples.

CHEMICALS INVOLVED

Dimethyl sulphate methylates guanine.

Acid removes any purines

Hydrazine modifies any pyrimidine

Hydrazine with NACL specifically modifies cytosines

Piperidine is used to remove the modified bases

SANGER METHOD

F. Sanger and his colleagues developed a second

method of DNA sequencing, which now is used
much more frequently than the Maxam-Gilbert method

The sequence of a single-stranded DNA molecule is

determined by enzymatic synthesis of complementary
polynucleotide chains, these chains terminating at specific
nucleotide positions

Sanger method is the most suitable method for

automation in large scale sequencing projects, and most
general sequencing is now carried out in this way

WHY SANGER METHOD TERMED AS

CHAIN TERMINATION METHOD????

Specific terminators of DNA Chain Elongation is

2’3’-dideoxy nucleoside triphosphates (ddNTPs)
can be incorporated normally into a growing DNA
chain through their 5’triphosphate groups

ddNTPs are telogens ,they can not form phospho -

diester bonds with the next incoming deoxynucleotides
(dNTPs).

Nucleotides which causes chain termination because they

lack 3’ hydroxyl group for extension .hence this technique
called as dideoxy (or) chain termination method.

CHAIN TERMINATION METHOD

PRINCIPLE

The single-stranded DNA to be sequenced serves as the

template strand for in vitro DNA synthesis; a synthetic
5′-end-labeled oligodeoxynucleotide is used as the primer

When a small amount of a specific dideoxy NTPs (ddNTPs)

is included along with the four deoxy NTPs normally
required in the reaction mixture for DNA polymerase

The products are the series of chains that are specifically

terminated at the dideoxy residue.

Thus four separate reactions, each containing a different

dideoxy NTP,can be run, and their products displayed on a
high-resolution Acryl amide gel.

CHAIN TERMINATION METHOD

PROCEDURE

Prepared the starting material for a chain termination sequencing experiment is a.
identical single-stranded DNA molecules.

To anneal a short oligonucleotide to the same position on each  molecule, this
oligonucleotide subsequently acting as the primer for synthesis of a new DNA
strand that is complementary  to the template catalyzed by DNA polymerase
requires  requires the four deoxyribonucleotide triphosphates (dNTPs - dATP,
dCTP, dGTP  and dTTP) as substrates, would normally continue  until several
thousand nuceotides had been polymerised.

The polymerase does not discriminate between dNTPs and ddNTPs, so the
dideoxynucleotide can be incorporated into the growing chain, but it then blocks
further elongation because it lacks the 3′-hydroxyl group needed to form a
connection with the nucleotide.

This process continue until several hundred nucleotides have been polymerized
before a ddATP is eventually incorporated. The result is therefore a set of new
chains, all of different lengths, but each ending in ddATP.

Now the polyacrylamide gel comes on to play. The family of the presence of

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Now the polyacrylamide gel comes on to play. The family of the presence of

ddNTPAs(ddATP,ddCTP,ddGTP,ddTTP) loaded into four adjacent wells of
the gel.

After electrophoresis, the DNA sequence can be read directly from the positions

of the bands in the gel.

HOW DOES THE DNA TEMPLATE OBTAINED?

The template for a chain termination experiment is a
single-stranded version of the DNA molecule to be
sequenced. There are several ways in which this can be
Obtained;

The DNA can be cloned in a plasmid vector.

The DNA can be cloned in a bacteriophage M13 vector.

The DNA can be cloned in a phagemid.

PCR can be used to generate single-stranded DNA.

THE DNA CAN BE CLONED IN A PLASMID VECTOR

The double stranded plasmid DNA strand must be converted

into single-stranded DNA by denaturation with alkali or by
boiling. This is a common method for obtaining template
DNA for DNA sequencing largely.

A single strand of plasmid is that it can be difficult to prepare

plasmid DNA that is not contaminated with small quantities

of bacterial DNA and RNA, which can act as spurious templates
or primers in the DNA sequencing experiment

THE DNA CAN BE CLONED IN A BACTERIOPHAGE M13

VECTOR

M13 bacteriophage has a single-stranded

DNA genome which,after infection of
Escherichia coli bacteria, is converted into a
double-stranded replicative form.

At the same time the infected cells
continually secrete new M13 phage
particles, approximately 1000 per
generation ,these phages containing the
single-stranded version of the genome.

The one disadvantage is that DNA

fragments longer than about 3 kb suffer
deletions and rearrangements when
cloned in an M13 vector, so the system
can only be used with short pieces of DNA.

PCR CAN BE USED TO GENERATE

SINGLE STRANDED DNA

One way of using PCR to prepare template DNA for chain

termination sequencing. The PCR is carried out with one
normal primer (shown in red), and one primer that is
labeled with a metallic bead (shown in brown). After PCR,
the labeled strands are purified with a magnetic device.

RECENT INNOVATIONS IN CHAIN

TERMINATION SEQUENCING

Thermal cycle sequencing

Automated DNA sequencing

Pyrosequencing

Sequencing by hybridization

THERMAL CYCLE SEQUENCING

( Sears et al., 1992)

The discovery of thermo stable DNA polymerases, which led to the

development of PCR has also resulted in new methodologies for chain
termination sequencing

Thermal cycle sequencing has two advantages over traditional chain

termination sequencing

1) uses double-stranded rather than single-stranded

DNA as the starting material.

2) very little template DNA is needed, so the DNA does

not have to be cloned before being sequenced

Thermal cycle sequencing is carried out in a similar way to PCR but just

one primer is used and each reaction mixture includes one of the

ddNTP. Because there is only one primer, only one of the strands of the
starting molecule is copied, and the product accumulates in a linear
fashion, not exponentially as is the case in a real PCR

The presence of the ddNTP in the reaction mixture causes chain

termination, as in the standard methodology, and the family of resulting
strands can be analyzed and the sequence read in the normal manner by
polyacrylamide gel electrophoresis

THERMAL CYCLE SEQUENCING

Thermal cycle sequencing.

PCR is carried out with just one primer and with a

dideoxynucleotide present in the reaction mixture. The result
is a family of chain-terminated strands - the ‘A' family in the
reaction shown. These strands, along with the products of the

C, G and T reactions, are electrophoresed as in the standard
methodology

AUTOMATED DNA SEQUENCING

(Leroy Hood and colleagues,1986)

The most dramatic advance in sequencing and the one that

carried DNA sequencing into a high throughput environment
was the introduction of automated sequencing using
fluorescence-labeled dideoxy-terminators.

In 1986, Leroy Hood and colleagues reported on a DNA

sequencing method in which the radioactive labels,
autoradiography, and manual base calling were all replaced
by fluorescent labels, laser induced fluorescence detection,

by fluorescent labels, laser induced fluorescence detection,
and computerized base calling.

In their method, the primer was labeled with one of four different

fluorescent dyes and each was placed in a separate sequencing
reaction with one of the four dideoxynucleotides plus all four
deoxynucleotides.

Fluorolabeling has been equally important in the development of

sequencing methodology, in particular because the detection system
for fluorolabels has opened the way to automated sequence reading.

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AUTOMATED DNA SEQUENCING WITH FLUORESCENTLY
LABELED DIDEOXYNUCLEOTIDES

(A)

The chain termination reactions are carried out in a

single tube, with each dideoxynucleotide labeled
with a different fluorophore.In the automated

sequencer, the bands in the electrophoresis gel
move past a fluorescence detector, which identifies

move past a fluorescence detector, which identifies

which dideoxynucleotide is present in each band.

The information is passed to the imaging system.

(B)

The printout from an automated sequencer.

The sequence is represented by a series of peaks,
one for each nucleotide position. In this example,

a green peak is an ‘A', blue is ‘C', black is ‘G',
and red is ‘T'.

PYROSEQUENCING

A novel DNA sequencing method in which addition of a nucleotide to the
end of a growing polynucleotide is detected directly by conversion of the
released pyrophosphate into a flash of chemiluminescence's

Does not require electrophoresis or any other fragment separation

procedure and so is more rapid than chain termination sequencing.

Template is copied in a straightforward manner without added ddNTPs.

During sequencing the addition of a nucleotide to the end of the growing

strand is detectable because it is accompanied by release of a molecule
of pyrophosphate, which can be converted by the enzyme sulfurylase
into a flash of chemiluminescence's.

Each dNTP is therefore added separately, one after the other, with a

nucleotidase enzyme also present in the reaction mixture so that if a
dNTP is not incorporated into the polynucleotide then it is rapidly
degraded before the next dNTP is added.

PYROSEQUENCING

The strand synthesis reaction is carried out in the
absence of dideoxynucleotides. Each dNTP is added

individually, along with a nucleotidase enzyme that

degrades the dNTP if it is not incorporated into the
strand being synthesized. Incorporation of a nucleotide

is detected by a flash of chemiluminescence induced
by the pyrophosphate released from the dNTP. The

order in which nucleotides are added to the growing

strand can therefore be followed.

SEQUENCED BY HYBRIDIZATION

The Hybridization technique is a very different approach to DNA

sequencing through the use of DNA chips might one day be possible.

A chip carrying an array of different oligonucleotides could be used in
DNA sequencing by applying the test molecule.

Hybridization to an individual oligonucleotide would indicate the

presence of that particular oligonucleotide sequence in the test
molecule, and comparison of all the oligonucleotides to which
hybridization occurs would enable the sequence of the test molecule
to be deduced.

The problem with this approach is that the maximum length of the

molecule that can be sequenced is given by the square root of the
number of oligonucleotides in the array.

SEQUENCED DY HYBRIDIZATION

The chip carries an array of every possible

8-mer oligonucleotide

The DNA to be sequenced is labeled with a

fluorescent marker and applied to the chip, and the
positions of hybridizing oligonucleotides determined
by confocal microscopy

Each hybridizing oligonucleotide represents an

8-nucleotide sequence motif that is present in the
probe DNA.

The sequence of the probe DNA can therefore be

deduced from the overlaps between the sequences
of these hybridizing oligonucleotides.