DNA SEQUENCING

OBJECTIVE

• To determine the nucleotide sequences present in the given DNA sample.

WHY??

To study the genomic organization of a organism

To identify the restriction sites in plasmids

Cloning

TECHNIQUES

Maxam Gilbert method

Sanger’s method

Other techniques

- Automated Sequencing

- Pyro sequencing

- Microarrays

Maxam-Gilbert method

• Allan Maxam – Walter Gilbert

• 1976

• Harward physist

• First developed sequencing method

• Chemical method

Requirement

DNA sample

NaOH

Chemicals

PAGE

Chemical Degradation of Purines

– Purines ( G) damaged by dimethylsulfate

– Methylation of base

– Heat releases base

– Alkali cleaves G

– Formic acid – modifies A,G

Chemical Degradation of Pyrimidines

– Pyrimidines (C, T) are damaged by hydrazine

– Piperidine cleaves the backbone

– 2 M NaCl inhibits the reaction with T

Chemical Reagents Used

Steps Involved

Denature the ds DNA with NaOH

5’ end of the single stranded DNA fragment to be sequenced is P-labelled

The labeled DNA fragment is then divided into four aliquots,

1. Aliquot A + dimethyl sulphate, which methylates guanine residue2. Aliquot B + formic acid, which modifies adenine and guanine residues3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific for cytosine

The four are incubated with piperidine which cleaves the sugar phosphate backbone of DNA next to the residue that has been modified.

Advantages

Homopolymeric DNA runs are sequenced as efficiently as heterogeneous

DNA sequences,

Used to analyze DNA-protein interactions (i.e., footprinting)

Used to analyze nucleic acid structure and epigenetic modifications to

DNA

Limitation

However, despite all improvements, these limitations remain:

(1) Gel electrophoresis is limited to 700-900 bp, with 400-500 bp more commonly attained

(2) The first 15-40 bp are often difficult to interpret

(3) Requires lots of purified DNA, and many intermediate purification steps

Disadvantages

This method is not commonly used today because:

(a) it requires extensive use of hazardous chemicals,

(b) it has a relatively complex set-up/technical complexity,

(c) it is difficult to "scale-up", and cannot be used to analyze more than 500 base pairs

Sangers method

• 1980

• Fred Sanger – protein chemist

• Nobel laureate

• Also,

Dideoxynucleotide method

Chain termination method

Enzymatic method

Dideoxy dNTP

Requirement

1. Single stranded template or target DNA

2. Sequence-specific primer

3. DNA polymerase

4. All 4 dNTP’s (dATP, dGTP, dCTP, dTTP)

5. All 4 ddNTP’s (ddATP, ddGTP, ddCTP, ddTTP)

Steps involved

- Template

- Primer (radio labeled)

- Polymerase 1

- Extension chemistry

- Termination (by ddNTP)

- Separation

- Detection

Problem

A cloned fragment of DNA was sequenced by using the dideoxy

method. A part of the autoradiogram of the sequencing gel is

represented here.

• Deduce the nucleotide sequence of the DNA nucleotide chain

synthesized from the primer. Label the 5′ and 3′ ends.

• Deduce the nucleotide sequence of the DNA nucleotide chain used

as the template strand. Label the 5′ and 3′ ends.

• Write the nucleotide sequence of the DNA double helix (label the 5′

and 3′ ends).

Comparison

• Sanger Method

– Enzymatic

– Requires DNA synthesis

– DNA is labeled

– Termination of chain

elongation

– Large base sequence

• Maxam Gilbert Method

– Chemical

– Requires DNA

– Primer is labeled

– Breaks DNA at different

nucleotides

– Small base sequence

Sample Output

1 lane

Summary

• Genetic information is stored in the order or sequence of nucleotides

in DNA.

• Chain termination sequencing is the standard method for the

determination of nucleotide sequence.

• Dideoxy-chain termination sequencing has been facilitated by the

the use of fluorescent dye detection.

• Alternative methods are used for special applications, such as

pyrosequencing (for resequencing and polymorphism detection) or

bisulfate sequencing (to analyze methylated DNA).

Thank you….,