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Dna Sequencing by Sanger Method

This document describes the Sanger sequencing method, including the materials and setup used. It explains how DNA replication occurs in the presence of both normal deoxynucleotide triphosphates (dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs), generating DNA fragments of different lengths. An example is provided of running a DNA sample with one of the nucleotide mixtures, showing the resulting band pattern on a gel, and asks what the band patterns from the other mixtures would look like.

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129 views19 pages

Dna Sequencing by Sanger Method

This document describes the Sanger sequencing method, including the materials and setup used. It explains how DNA replication occurs in the presence of both normal deoxynucleotide triphosphates (dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs), generating DNA fragments of different lengths. An example is provided of running a DNA sample with one of the nucleotide mixtures, showing the resulting band pattern on a gel, and asks what the band patterns from the other mixtures would look like.

Uploaded by

Sethu SK
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Introduction

to
Bioinformatics
BIN101

DNA Sequencing by Sangers Method


Sanger Sequencing Materials
 DNA Polymerase
 Primer (radioactive)
 Template DNA
 dNTPs (deoxyNTP)
 ddNTPs
(dideoxyNTP)
Sanger Sequencing Setup

 What do you think will happen if you allow DNA


replication to occur in the presence of both
deoxyATP and dideoxyATP?

Tube A
15% ATP
25% GTP
25% TTP
25% CTP
10% ddATP
Sanger Sequencing Setup

AAT C T G G G C TA C T C G G G C G T T
C G CA

Tube A
15% ATP
25% GTP
25% TTP
25% CTP
10% ddATP
Sanger
Sequencing
AAT C T G G G C TA C T C G G G C G T T
C G CA

Setup A G C C C G CA
Tube A
15% ATP
25% GTP
25% TTP
25% CTP
10% ddATP
Sanger
Sequencing
AAT C T G G G C TA C T C G G G C G T T
C G CA

Setup A G C C C G CA

AT G A G C C C G CA
Tube A
15% ATP
25% GTP
25% TTP
25% CTP
10% ddATP
Sanger
Sequencing
AAT C T G G G C TA C T C G G G C G T T
C G CA

Setup A G C C C G CA

AT G A G C C C G CA
Tube A
15% ATP
25% GTP
AC C C GATGAG C C C
GGCC
GAT
25% TTP
25% CTP
10% ddATP
Sanger
Sequencing
AAT C T G G G C TA C T C G G G C G T T
C G CA

Setup A G C C C G CA

AT G A G C C C G CA
Tube A
15% ATP
25% GTP
AC C C GATGAG C C C
GGCC
GAT
25% TTP
A G A C C C G A T G A G C C C G CA 25% CTP
10% ddATP
Problem:
1. The following DNA fragment was sequenced by the Sanger method. The red asterisk
indicates a fluorescent label.

A sample of the DNA was reacted with DNA polymerase and each of the nucleotide
mixtures (in an appropriate buffer) listed below. Dideoxynucleotides (ddNTPs) were added
in relatively small amounts.
1. dATP, dTTP, dCTP, dGTP, ddTTP
2. dATP, dTTP, dCTP, dGTP, ddGTP
3. dATP, dCTP, dGTP, ddTTP
4. dATP, dTTP, dCTP, dGTP
The resulting DNA was separated by electrophoresis on an agarose gel, and the fluorescent
bands on the gel were located. The band pattern resulting from nucleotide mixture
1 is shown below.
Assuming that all mixtures were
run on the same gel,
what did the remaining lanes
of the gel look like?

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