plik

DNA sequencing by the Sanger

method

The standard DNA sequencing technique is the Sanger method,
named for its developer, Frederick Sanger, who shared the 1980
Nobel Prize in Chemistry. This method begins with the use of
special enzymes to synthesize fragments of DNA that terminate
when a selected base appears in the stretch of DNA being
sequenced. These fragments are then sorted according to size

sequenced. These fragments are then sorted according to size
by placing them in a slab of polymeric gel and applying an
electric field -- a technique called electrophoresis. Because of
DNA's negative charge, the fragments move across the gel toward
the positive electrode. The shorter the fragment, the faster it
moves. Typically, each of the terminating bases within the
collection of fragments is tagged with a radioactive probe for
identification.

DNA sequencing example

Problem Statement: Consider the following DNA
sequence (from firefly luciferase). Draw the sequencing
gel pattern that forms as a result of sequencing the
following template DNA with ddNTP as the capper.

atgaccatgattacg...

Solution:

Given DNA template: 5'-atgaccatgattacg...-3'

DNA synthesized: 3'-tactggtactaatgc...-5'

DNA sequencing example

Given DNA template: 5'-atgaccatgattacg...-3'

DNA synthesized: 3'-tactggtactaatgc...-5'

Gel pattern:

+-------------------------+

lane ddATP

| W | | ||

lane ddTTP

| W | | | | |

lane ddCTP

| W | | |

lane ddCTP

| W | | |

lane ddGTP

| W || |

+-------------------------+

Electric Field +
Decreasing size

where "W" indicates the well position, and "|"

denotes the DNA bands on the sequencing gel.

A sequencing gel

This picture is a radiograph. The dark color of the lines is
proportional to the radioactivity from

P labeled adenonsine

in the transcribed DNA sample.

Reading a sequencing gel

You begin at the right, which are the smallest DNA fragments.
The sequence that you read will be in the 5'-3' direction.
This sequence will be exactly the same as the RNA that
would be generated to encode a protein. The difference is that
the T bases in DNA will be replaced by U residues. As an example,
in the problem given, the smallest DNA fragment on the sequencing
gel is in the C lane, so the first base is a C. The next largest band

gel is in the C lane, so the first base is a C. The next largest band
is in the G lane, so the DNA fragment of length 2 ends in G.
Therefore the sequence of the first two bases is CG.
The sequence of the first 30 or so bases of the DNA are:

CGTAATCATGGTCATATGAAGCTGGGCCGGGCCGTGC....

When this is made as RNA, its sequence would be:

CGUAAUCATGGUCAUAUGAAGCUGGGCCGGGCCGUGC....

Note that the information content is the same, only the T's have
been replaced by U's!.

The codon table

5’-Base

Middle Base

3’-Base

U(=T)

U(=T)

Phe

Ser

Tyr

Cys

U(=T)

Phe

Ser

Tyr

Cys

Leu

Ser

Term

Leu

Ser

Term

Trp

Leu

Pro

His

Arg

U(=T)

Leu

Pro

His

Arg

Leu

Pro

His

Arg

Leu

Pro

Gln

Arg

Leu

Pro

Gln

Arg

Ile

Thr

Asn

Ser

U(=T)

Ile

Thr

Asn

Ser

Ile

Thr

Lys

Arg

Met

Thr

Lys

Arg

Val

Ala

Asp

Gly

U(=T)

Val

Ala

Asp

Gly

Val

Ala

Glu

Gly

Val

Ala

Glu

Gly

Translating the DNA

sequence

The order of amino acids in any protein is specificed by the
order of nucleotide bases in the DNA.
Each amino acid is coded by the particular sequence of three bases.
To convert a DNA sequence

First, find the starting codon. The starting codon is always

the codon for the amino acid methionine. This codon is

AUG in the RNA (or ATG in the DNA):

GCGCGGGUCCGGGCAUGAAGCUGGGCCGGGCCGUGC....

Met

In this particular example the next codon is AAG. The first base
(5'end) is A, so that selects the 3rd major row of the table. The
second base (middle base) is A, so that selects the 3rd column of
the table. The last base of the codon is G, selecting the last line in
the block of four.

Translating the DNA

sequence

This entry AAG in the table is Lysine (Lys).
Therefore the second amino acid is Lysine.

The first few residues, and their DNA sequence, are as follows

(color coded to indicate the correct location in the

codon table):

Met Lys Leu Gly Arg … ...

AUG AAG CUG GGC CGG GCC GUG C..

This procedure is exactly what cells do when they synthesize
proteins based on the mRNA sequence. The process of translation
in cells occurs in a large complex called the ribosome.

Automated procedure for DNA

sequencing

A computer read-out of the gel generates a “false color” image
where each color corresponds to a base. Then the intensities are
translated into peaks that represent the sequence.

High-throughput seqeuncing:

Capillary electrophoresis

The human genome project
has spurred an effort to
develop faster, higher
throughput, and less
expensive technologies

Laser

Focusing

lens

Sheath flow cuvette

Sheath flow

expensive technologies
for DNA sequencing.
Capillary electrophoresis
(CE) separation has many
advantages over slab gel
separations. CE separations are faster and are capable of producing
greater resolution. CE instruments can use tens and even
hundreds of capillaries simultaneously. The figure show a simple
CE setup where the fluorescently-labeled DNA is detected as it
exits the capillary.

PMT

Collection Lensc

Beam block

filter

Sieving matrix for CE

It is not easy to analyze DNA in capillaries filled only with
buffer. That is because DNA fragments of different lengths
have the same charge to mass ratio. To separate DNA fragments
of different sizes the capillary needs to be filled with sieving
matrix, such as linear polyacrylamide (acrylamide polymerized
without bis-acrylamide).This material is not rigid like a cross-

without bis-acrylamide).This material is not rigid like a cross-
linked gel but looks much like glycerol. With a little bit of
effort it can be pumped in and out of the capillaries. To simulate
the separation characteristics of an agarose gel one can use
hydroxyethylcellulose. It is not much more viscous then water
and can easily be pumped into the capilliaries.

Fluorescent end labeling of

DNA