DNA sequencing by the Sanger method

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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
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.
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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'
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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 ddGTP W

-------------------------
Electric Field

Decreasing size where "W" indicates the well
position, and "" denotes the DNA bands on the
sequencing gel.
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A sequencing gel
This picture is a radiograph. The dark color of
the lines is proportional to the radioactivity
from 32P labeled adenonsine in the transcribed
DNA sample.
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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 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!.
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The codon table
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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.
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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.
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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.
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High-throughput seqeuncingCapillary
electrophoresis
The human genome project has spurred an effort
to develop faster, higher throughput, and less
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.
Sheath flow
Laser
Focusing lens
Sheath flow cuvette
Beam block
Collection Lensc
Collection Lensc
PMT
filter
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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- 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.
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Fluorescent end labeling of DNA