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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

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dna sequencing by the sanger method
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


dna sequencing example
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.



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

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

dna sequencing example3
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.

a sequencing gel
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.

reading a sequencing gel
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:


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


Note that the information content is the same, only the T's have

been replaced by U's!.

translating the dna sequence
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):



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 sequence8
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 … ...


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
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
High-throughput seqeuncing:Capillary 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




Sheath flow cuvette

Beam block

Collection Lensc

Collection Lensc



sieving matrix for ce
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.