DNA SEQUENCING

DNA sequencing is the process of determining the precise order of

nucleotide within a DNA molecule.

It includes any method or technology that is used to determine the

order of the four bases :-Adenine,Guanine,Cytosine,Thymine in a
strand of Dna

The advent of rapid DNA sequencing methods greatly accelerated

biological and medical research &Discovery

USE :-
DNA sequencing may be used to determine the sequence of
individual genes ,larger genetic region ,full chromosomes or entire
genomes.

Depending on the methods used,sequencing may provide the order

of nucleotides in DNA or RNA isolated from cells of
Anima,plants ,bacteria or NY other source of genetic information.

KNOWLEDGE OF DNA SEQUENCE HAS BECOME

INDISPENSABLE FOR BASIC BIOLOGICAL RESEARCH &IN
 NUMEROUS APPLIED FIELDS SUCH AS DIAGNOSTIC ,
BIOTECHNOLOGY ,FORENSIC BIOLOGY AND
BIOLOGICAL SYSTEMATICS.

HISTORY
Developed by Allan madam and Walter gilbert.
1st time analysis the DNA sequencing in 0(phi) in 1977.
BASIC METODS:-

1. maxam-gilbert dna sequencing

2.CHAIN TERMINATION OR DIDEOXY METHOD BY

SANGER

3.SHOTGUN SEQUENCE METHOD

4. 2ND GENERATION SEQUENCE METHOD

* PYROSEQUENCING

MAXAM -GILBERT SEQUENCING

 MAXAM GILBERT SEQUENCING REQUIRE
RADIOACTIVE LABELING AT ONE 5’END OF THE
DNA & PURIFICATION OF THE DNA FRAGMENTS TO
BE SEQUENCED.

CHEMICAL TREATMENT:-
* THE PURINES (A+G) ARE DEPURINATED USING
FORMIC ACID .
*THE GUANINES ARE METHYLATED BY DIMETHYL
SULPHATE.
*THE PYRIMIDINE (C+T) ARE METHYLATED USING
HYDRAZINE.
*THE ADDITION OF SALT TO HYDRAZINE
REACTION INHIBITS THE METHYLATION OF
THYMINE FOR THE C-REACTION.
THE MODIFIED DNA MAY THAN BE CLEAVED BY
HOT PIPERDINE AT THE POSTION OF THE
MODIFIED BASE.RUN FOR SEPARATE REACTIONS
EACH WITH DIFFERENT DDNTPS. RUN A GEL IN
FOUR SEPARATES LANES.
 SANGER OR DIDEOXY METHOD
1denaturatin
2.Primer attachment and extension
3.Termination
4.Gel electrophoresis

*the ddntps may be radioactively labelled with 32atp or

fluoroscently labelled with a fluoroscent dye for detection
inautomated sequencing machine.

THE DNA BANDS MAY THAN BE VISUALIZED BY

AUTORADIOGRAPHY OR UV LIGHT

*BPYROSEQUENCING :- PYROSEQUENCING IS A
METHOD OF A DNA SEQUENCIN THAT DETECTS
 LIGHT EMITTED DURING THE SEQUENTIAL
ADDITION OF NUCLEOTIDE DURING THE
SYNTHESIS OF A COMPLEMENTARY STRAND OF
DNA.IT DIFFERS FROM SANGER METHOD.
 PYROSEQUENCING METHOD IS BASED ON
DETECTING THE ACTIVITY OF DNA
POLYMERASE WITH ANOTER
CEMILUMINESCENT ENZYME.
 THE LIGHT PRODUCED IN THE LUCIFERASE-
CATALYZED REACTION IS DETECTED BY A
CAMERA AND ANALYZED IN A PROGRAM.

1ST METHOD -SOLID PHASE-IN SOLID

PYROSEQUENCING DON’T USE
ELECTROPHORESIS.
 3ENZYME
 WASH STEP TO REMOVE NUCLEOTIDEAFTER
EACH ADDITION.

2nd method -liquid phase-

3 enzyme+apyrase
Eliminate need for washing step.

DISADVANTAGES-
Smaller sequence can be sequenced
It can only sequence 300-500nucleotide
Pyrosequencing is most commonly used for sequencing or
resequencing of genomes for which the sequence ofa close relative is
already available.
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