This document discusses DNA sequencing methods. It describes the Maxam-Gilbert method, which uses chemical degradation of DNA to determine nucleotide sequences. It also describes the Sanger method, the most commonly used modern method, which uses DNA polymerase and dideoxynucleotides to terminate DNA strand elongation at specific positions. The document compares the two main historical sequencing methods and discusses their principles, steps, advantages, and disadvantages.

2. It is a technique used to
determine the order of
nucleotides (i.e. A, T, C, G) in a
strand of DNA.

3. Gene isolation
Sequence characterization
Forensics
Gene-Gene Interaction
Gene-Protein Interaction
Cloning
NEED FOR DNA SEQUENCING

4. DNA SEQUENCING METHOD
o Sequencing method became available in late 1970’s.
o Historically there are two methods are used:
1. Maxam and Gilbert method
2. Sanger method
o Modern sequencing method used principle of Sanger
method.

5. MAXAM and GILBERT METHOD
• It was given by A. Maxam and W. Gilbert in
1977.
• It involves chemical degradation of original
DNA rather then synthesis.
Principle: The sequence of dsDNA molecule is
determine by treatment of chemical that cut
the molecule at specific nucleotide position.

6. STEPES
 Denature a double – stranded DNA to single – stranded
by increasing temperature .
 Then ssDNA that is labelled at one end (either its 5’ or
3’ end) with radioactive P32 using either polynucleotide
kinase or terminal transferase.
 Cleave DNA strand at specific positions using chemical
reaction.
 G: Dimethylsulphate and piperidine
 A : Dimethylsulphate, an acid and piperidine
 T : Hydrazine and piperidine.
 C : Hydrazine, NaCl and piperidine.

7. Contd...
 Divide the mixture in four samples, each treating with different
reagent having property of destroying either only G or only C or A-
G or T-C
 Fragments are electrophoreses in high resolution acrylamide gel
for size separation These gels are placed under X-ray film, which
then yield a series of dark bands which show the location of
radiolabel DNA molecules.
 The fragment ordered by size and so we can deduce the sequence
of the DNA molecule.

9. DISADVENTAGES
This process is not much popular, due to
 time consuming
Labour intensive
Radio labelled elements are harmful
Chemicals are carcinogenic

10. • It was given by Frederick Sanger et al. In 1977.
• It is also known as dideoxynucleotide chain
termination method because
dideoxynucleotide are used as chain
terminator to produce a ladder of molecule.

11. Principle: A label primer is used to initiate
DNA synthesis ,and the addition of 4 different
dideoxynucleotides randomly arrest synthesis of
DNA.
Requirements:
• Single strand DNA template
• A primer with free 3’-OH end to start DNA
synthesis
• DNA Polymerase
• ddNTP(dideoxynucleotide Triphodphate).

12. STEPS -
 DNA strands are separated.
 The radioactive primer bind to the 3’ end of the fragment.
 DNA polymerase is added which synthesizes a complimentary DNA
sequencing.
 Every time a specific ddNTP is used in the complimentary strand , the
DNA synthesis halt.
 This creates fragments of different lengths, depend on the position of
incorporation of ddNTP.
 The fragment of each tube are separated by electrophoreses of high
resolution of Polyacrylamide Gel.

14. Sanger method
• Enzymatic
• Requires DNA sequence
• Termination of chain
elongation
• Automation
• Single strand DNA
Maxam Gilbert method
• Chemical
• Requires DNA
• Breaks DNA at different
nucleotides
• Automation is not
available
• Double stranded or
single stranded