Slide 1: DNA
SEQUENCING Presented By:-
B.Tech+M.Tech(Biotech). Why DNA is sequenced ? : Why DNA is sequenced ? To determine the order of nucleotide in a given DNA molecule. FOUNDERS OF SEQUENCING TECHNOLOGY : FOUNDERS OF SEQUENCING TECHNOLOGY Sanger Wally Gilbert Various techniques for DNA sequencing : Various techniques for DNA sequencing Historically there are two main methods of DNA sequencing:
MAXAM and GILBERT method
Other alternative methods: -
Automated DNA sequencing
Gene chips (microarrays) etc. MAXAM and GILBERT method (chemical degradation method) : MAXAM and GILBERT method (chemical degradation method) Allan Maxam & Walter Gilbert developed chemical degradation technique for DNA sequencing.
This method is based on the chemical modification of DNA & subsequent cleavage at specific bases. AT ATCGAT Specific Reaction to G Chemical
(invisible) Requirements:- : Requirements:- Double stranded DNA.
Dimethyl sulphate methylates guanine.
Hydrazine modifies any pyrimidine.
Hydrazine with Nacl specifically modifies cytosine.
Piperidine is used to cleave phosphodiester bond just after modify nucleotide. Slide 7: Procedure Double stranded DNA fragment is labelled by attaching radioactive phosphorus group to 5’ en Double stranded DNA fragment is labeled by attaching radioactive phosphorus group to 5’ end. Denature the DNA by heating at above 900C or alkali treatment. Electrophoresis is done to seprate 2 strands on the basis that one strand contain more purine nucleotides than other, so one strand is slightly heavier and runs slowly. next Slide 8: one strand is purified from gel and divide into 4 tubes. Modify A
piperidine Modify T
piperidine Modify G
piperidine Modify C
piperidine Cleaved fragment retain phosphate label at their 5’end. Polyacrylamide gel electrophoresis & after it visualize by autoradiography. The sequence is read from the bottom to top of the gel. Slide 9: Fig: Maxam method SANGER’S METHOD (CHAIN TERMINATION METHOD) : SANGER’S METHOD (CHAIN TERMINATION METHOD) Fred Sanger & A.R.Coulson(1977) developed chain termination method.
This method based on the principle that the replacement of dNTPs with ddNTPs in DNA chains which terminates DNA synthesis. A Terminated Biosynthetic method Template or Producing various fragments Requirements:- : Requirements:- Single stranded DNA which is to be sequenced.
M13 as a vector.
A suitable primer (a small piece of DNA that can pair with the template DNA to act as a starting point for replication).
DNA polymerase (an enzyme that adding new nucleotides to the 3’ end of the template).
A small proportion of radiolabeled dideoxynucleotides (ddNTPs). Dideoxy Nucleotides : Dideoxy Nucleotides A dideoxynucelotide is a molecule that lacks a hydroxyl group at both the 2’ and 3’ carbons of the sugar.
These are resemble to normal nucleotides but lack the normal -OH group which causes chain termination. Slide 13: Phosphodiester
bond 3’ 5’ dideoxynuceotide Terminated ddNTP Normal Linking Can not react Procedure : DNA that is to be sequenced is mixed with a primer and dNTPs.
Radiolabeled dideoxynucleotides are added saperately to each of the four tubes. Procedure Next M13 acts as template for primer DNA area to be sequenced Slide 15: Add
DNA polymerase Add
DNA polymerase Add
DNA polymerase Add
DNA polymerase Terminate fragment retain radiolabel ddNTP at their 3’end. Polyacrylamide gel electrophoresis & after it visualize by autoradiography. Next Gels are made up of 6-10% polyacrylamide which contain urea.Electrophoresis sepration takes place in 3-10 hours. : Sequencing gels are read from bottom to top (5′ to 3′). G A T C 3′
5′ Gels are made up of 6-10% polyacrylamide which contain urea.Electrophoresis sepration takes place in 3-10 hours. Slide 17: Fig: Sanger’s method REFERENCES: : REFERENCES: Gene Cloning and DNA analysis; T.A Brown (Fifth edition)
Principles of gene manipulation and genomics; S.B. Primrose and R.M Twyman( Seventh edition). Slide 20: QUERIES ???