DNA Sequencing Technique

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