6.DNA Sequencing
6.DNA Sequencing
6.DNA Sequencing
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P 5′ G A C G T G C A A C G A A 3′
Chemical Modification and Cleavage
• Base Modification using Dimethyl sulphate
– Purine
• Adenine
• Guanine
– Only DMS------- G
– DMS+ Formic acid-------G+A
DMS FA H H+S
G G C C
G A T C
G G T
G G C C
C
A T
G C
A C
A T
P 5′ G A C G T G C A A C G A 3′
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Maxam And Gilbert Sequencing:
10 nucleotide DNA sequence:
5’P-TTCAGCCGAT-OH3’
First step:
5’P-TTCAGCCGAT-OH3’+H2O 5’OH-TTCAGCCGAT-OH3’+Pi
2. A+G :
- Here the DNA is protonated rather than methylated.
- TTC, TTCA, TTCAGCC, TTCAGCCG.
- measuring 3,4 ,7 and 8 nucleotides in length.
3. C+T :
- DNA is reacted with hydrazine(NH2-NH2) and this followed with
piperidine treatment.
- T, TT, TTCAG, TTCAGC, TTCAGCCGA.
- measuring 1, 2, 5, 6 and 9 nucleotide in length
4. C only :
– STEP 1: Denaturation
– Step 3: Termination
ddCTP + dAdGddC
C
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC
ddGTP + dAddG
G four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG
T
ddTTP + dAdGdCddT
four dNTPs dAdGdCdTdGdCdCdCdG
Chain Termination Sequencing
3′
G
G A T C G
Longer fragments T
A
ddG
A
A
T
C
Shorter fragments A
ddG T
G
5′
3’-ATGTGCTAGCT-5’
3’-ATGTGCTAGCT-5’
5’-TA-3’
5’-T-3’ 5’-TACA-3’
5’-TACACGAT-3’ 5’-TACACGA-3’
5’-TACACGATCGA-3’
Amplification in dGTTP Amplification in ddCTP
3’-ATGTGCTAGCT-5’ 3’-ATGTGCTAGCT-5’
5’-TACACG-3’ 5’-TAC-3’
5’-TACACGATCG-3’ 5’-TACAC-3’
5’-TACACGATC-3’
Reading Sequence
BAND ddTTP ddATP ddGTP ddCTP 3’ 3’
12 bp
11 bp
10 bp
9 bp
8 bp
7 bp
6 bp
5 bp
4 bp
3 bp
2 bp
1 bp
5’ 5’
Sanger Sequencing: Process Summarized
• DNA polymerase
• ATP sulfurylase
• Luciferase and apyrase
• Substrates adenosine 5´ phosphosulfate (APS) and luciferin
Applications of dna sequencing:
• To Find Genes
• Information regarding MUTATIONS
• Gene overlapping
• Identification of POLYMORPHSIMS
• Profiling of the DNA methylation in the genomes
• Exome sequencing
• Identification of GENE REGULATORY CONTROL
SEQUENCES
• To determine the PATERNITY of a child
REFERENCES:
• text book of biochemistry by d.m.vasudevan
• Bio physical chemistry by upadhyaya and nath
• tietz textbook of clinical chemistry
• molecular biology— burtan .e. tropp
• Wilson and Walker Principles and techniques of Biochemistry
• Cell and molecular biology– e.d.p.de Roberti's 8 th edition
• Bioinformatics– practical approach– shui Qing ye
• Textbook of Biochemistry by u SatyaNarayana
• http://en.wikipedia.org/wiki/DNA_sequencing --Pyro sequencing
• DNA sequencing Written by: Anthony J.F. Griffiths