DNA sequencing.pdf

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.

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 32 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

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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