DNA Sequencing : Maxam Gilbert and Sanger Sequencing
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DNA SEQUENCINGChemical Modification Method
Chain Termination MethodVeerendra Singh Nagoria
Assistant Professor Biotechnology
Objectives
• What is DNA Sequencing ?
• History of development
• Basic Methods- Chain
termination and Chemical
modification method
What is DNA Sequencing ?
• Determining the precise order of nucleotides in DNA.
• We need to determine the order of nucleotide bases in a strand of DNA for sequencing.
The Need for DNA Sequencing
• Gene isolation• Sequence charaterization• Forensics• Molecular Archeology• Gene Gene Interaction• Gene Protein Interaction• Cloning
DNA • Deoxyribonucleic Acid• Stores genetic information• Four different nucleotides A,T,G,C• DNA comprises of a long molecule analogous to a chain,
while the links of the chain are called Nucleotides
Historical Timeline
1870 – Miescher discovers DNA
1940 - Avery: Proposes DNA as ‘Genetic Material’
1953 – Watson & Crick “double helical structure”
1970 - Wu: Sequences λ Cohesive End DNA
1977 – Sanger: Dideoxy Chain Termination
1977 – Gilbert: Chemical Degradation
1986 – Partial Automation
1990 – Cycle Sequencing, Improved Sequencing Enzymes,
Improved fluorescent detection schemes
2002 – NGS: 454 , pyro sequencing
Cost per Genome
Cost per Megabases
Cost Data (source NHGRI)
Date Cost per Mb Cost per Genome Sep-01 $5,292.39 $95,263,072Mar-02 $3,898.64 $70,175,437Sep-02 $3,413.80 $61,448,422Mar-03 $2,986.20 $53,751,684Oct-03 $2,230.98 $40,157,554Jan-04 $1,598.91 $28,780,376Apr-04 $1,135.70 $20,442,576Jul-04 $1,107.46 $19,934,346Oct-04 $1,028.85 $18,519,312Jan-05 $974.16 $17,534,970Apr-05 $897.76 $16,159,699Jul-05 $898.90 $16,180,224Oct-05 $766.73 $13,801,124Jan-06 $699.20 $12,585,659Apr-06 $651.81 $11,732,535Jul-06 $636.41 $11,455,315Oct-06 $581.92 $10,474,556Jan-07 $522.71 $9,408,739Apr-07 $502.61 $9,047,003Jul-07 $495.96 $8,927,342Oct-07 $397.09 $7,147,571Jan-08 $102.13 $3,063,820Apr-08 $15.03 $1,352,982
Date Cost per Mb Cost per Genome Oct-08 $3.81 $342,502Jan-09 $2.59 $232,735Apr-09 $1.72 $154,714Jul-09 $1.20 $108,065Oct-09 $0.78 $70,333Jan-10 $0.52 $46,774Apr-10 $0.35 $31,512Jul-10 $0.35 $31,125Oct-10 $0.32 $29,092Jan-11 $0.23 $20,963Apr-11 $0.19 $16,712Jul-11 $0.12 $10,497Oct-11 $0.09 $7,743Jan-12 $0.09 $7,666Apr-12 $0.07 $5,901Jul-12 $0.07 $5,985Oct-12 $0.07 $6,618Jan-13 $0.06 $5,671Apr-13 $0.06 $5,826Jul-13 $0.06 $5,550Oct-13 $0.06 $5,096Jan-14 $0.04 $4,008Apr-14 $0.05 $4,920
Sequencing Methods• To determine the order of the nucleotide
bases adenine, guanine, cytosine, and thymine in a molecule of DNA two methods were used1. Maxam and Gilbert; Chemical Sequencing2. Sanger; Chain Termination Sequencing
• These two are conventional methods• Robotics and automated sequencing are
based on these methods
Maxam and Gilbert Method• In 1976–1977, Allan Maxam and Walter Gilbert
developed a DNA sequencing method based on chemical modification of DNA and subsequent cleavage at specific bases
I. Chemical Modification of DNA; radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma-32P ATP)
II. Purification of the DNA fragment to be sequencedIII. Chemical treatment generates breaks in DNAIV. Run on the gel
Chemical Modification and Cleavage
• Ploy nucleotide Kinase radioactive label at one 5' end of the DNA using gamma-32P
5 ′ G A C G T G C A A C G A A 3′
32P 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
• Cleavage of Sugar Phosphate backbone using Piperidine
Chemical Modification and Cleavage
• Base modification using Hydrazine– Pyrimidine• Cytocine• Thymidine
– Hydrazine----- C+T– Hydrazine + NaCl--------C
• Cleavage of Sugar Phosphate backbone using Piperidine
DMS
G
GG
G
FA
GA
GG
AG
AA
H
CTT
C
TC
CT
H+S
CC
CC
Maxam Gilbert Sequencing
32P 5 ′ G A C G T G C A A C G A 3′
Sequencing gels are read from bottom to top (5 to 3 ).′ ′
G G+A T+C C
3′AGCAACGTGCAG5′
Longer fragments
Shortest fragments
G
A
Maxam-Gilbert Sequencing
32P 5 ′ G A C G T G C A A C G A 3′
Maxam Gilbert Sequencing: Process Summarized
1. Label 5’- end of DNA 2. Aliqot DNA sample in 4 tubes3. Perform base modification reaction4. Perform Cleavage reaction5. Perform Gel Electrophoresis6. Perform Autoradiography7. Interpret results
Sanger; Chain Termination Sequencing
• It is PCR based method• A modified DNA replication reaction• Growing chains are terminated by
dideoxynucleotides
The 3 -OH group necessary for formation of the phosphodiester bond is missing in ddNTPs′
ddATP + ddAfour dNTPs dAdGdCdTdGdCdCdCdG
ddCTP + dAdGddCfour dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC dAdGdCdTdGdCdCddC
ddGTP + dAddGfour dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG
ddTTP + dAdGdCddTfour dNTPs dAdGdCdTdGdCdCdCdG
A
C
G
T
Sanger; Chain Termination Sequencing
A G C T G C C C G
Sequencing gels are read from bottom to top (5 to 3 )′ ′
G A T C
3′GGTAAATCATG5′
Longer fragments
Shorter fragmentsddG
ddG
Chain Termination Sequencing
Sanger Sequencing: An Example
5’-TACACGATCGA-3’3’-ATGTGCTAGCT-5’
Denature the sequenceUse only forward primer i.e. using 3’-5
3’-ATGTGCTAGCT-5’5’-T-3’5’-TACACGAT-3’
Amplification in ddTTP
3’-ATGTGCTAGCT-5’5’-TA-3’5’-TACA-3’5’-TACACGA-3’5’-TACACGATCGA-3’
Amplification in ddATP
Amplification in dGTTP Amplification in ddCTP
3’-ATGTGCTAGCT-5’5’-TACACG-3’5’-TACACGATCG-3’
3’-ATGTGCTAGCT-5’5’-TAC-3’5’-TACAC-3’5’-TACACGATC-3’
Reading SequenceBAND ddATP ddTTP ddGTP ddCTP12 bp
11 bp
10 bp
9 bp
8 bp
7 bp
6 bp
5 bp
4 bp
3 bp
2 bp
1 bp
3’
5’ 5’
3’
Sanger Sequencing: Process Summarized
1. Get enough quantity of DNA (Run PCR)2. Aliqot DNA into four different tubes3. Prepare PCR reaction mix as below:• Primer, taq PM, template(ss DNA), dNTPS (All)
and ddNTPs(ddATP, ddGTP,ddCTP & ddTTP respectively)
4. Run PCR5. Perform Gel Electrophoresis6. Interpret results
Thank You
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