Sanger & maxam gilbert sequencing @ujjwalsirohi

sanger sequencing (chain termination method)
maxam gilbert (chain degradation method)
Basic methods of sequencing..

1. @ujjwalsirohi
PhD scholar

2. content
Sanger sequencing
MaxamGilbert Sequencing

3. Sanger sequencing
Based on the selective incorporation of chain terminating
dideoxynucleotidesbyDNA polymeraseduringin
vitroDNA replication.
Developed byFrederick Sangerand colleagues in 1977.
99.9% accuracy.
Cost: $1,000 per million bp.
In 1990 ~450bp (blotting system)
In 2017 ~1100bp can be sequenced (ABI 3730)

5. Method
The sequence of a single-stranded DNA molecule is
determined by enzymatic synthesis of
complementary polynucleotide chains.
These chains terminating at specific nucleotide
positions.
Separate by gel electrophoresis.
Read DNA sequence.

6. 3’TGAGTCTACGA5’ (to be sequenced)

8. Maxam–Gilbert sequencing
Developed byAllan MaxamandWalter
Gilbertin 1976–1977.
This method is based onnucleobase-specific
partial chemical modification of DNA and
subsequentcleavageof the DNA backbone at sites
adjacent to the modifiednucleotides.

9. methodology
Denature a double-stranded DNA to single-stranded by
increasing temperature.
Radioactively label one 5' end of the DNA fragment to be
sequenced by a kinasereaction using gamma-32 P.
Cleave DNA strand at specific positions using chemical
reactions.
The chemical treatment cleaves at G, A+G, C and C+T.
A+G means that it cleaves at A, but occasionally at G as
well.
In four reaction tubes, we will have several differently sized
DNA strands.

10. Dimethyl
sulphate(DM
S)
Pepridine
formate
(pH2)
Hydrazine Hydrazine +
NaCl
Methylation
(G)
Weakens
glycosidicbond
of purines(G+A)
Splits the ring of
pyrimidine
(T+C)
Only reacts on
cytosine (C)
Piperidinetreatment (at 90˚C) to cleave sugar-P chain
electrophoresis