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
[Link] TERMINATION OR DIDEOXY METHOD BY
SANGER
[Link] 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 [Link] FOR SEPARATE REACTIONS
EACH WITH DIFFERENT DDNTPS. RUN A GEL IN
FOUR SEPARATES LANES.
SANGER OR DIDEOXY METHOD
1denaturatin
[Link] attachment and extension
[Link]
[Link] 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
[Link] 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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