DNA sequencing is a technique to find out the exact arrangement of Nucleotides to make one strand of DNA. DNA sequencing helps in numerous ways from sequence information to paternity testing, mutation detection etc. Traditionally two approaches were used to solve the problem. First is based of enzymes and Second is based on ddNTPs to sequence the DNA using gel electrophoresis technique.

1. DNA
SEQUENCINGChemical Modification Method
Chain Termination Method
Veerendra Singh Nagoria
Assistant Professor
Biotechnology

2. ObjectivesObjectives
• What is DNA Sequencing ?
• History of development
• Basic Methods- Chain
termination and Chemical
modification method

3. What isDNA Sequencing ?What isDNA 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.

4. The Need for DNA SequencingThe Need for DNA Sequencing
• Gene isolation
• Sequence charaterization
• Forensics
• Molecular Archeology
• Gene Gene Interaction
• Gene Protein Interaction
• Cloning

5. DNADNA
• 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

6. Historical TimelineHistorical 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

7. Cost per GenomeCost per Genome

8. Cost per MegabasesCost per Megabases

9. Cost Data (source NHGRI)Cost Data (source NHGRI)
Date Cost per Mb Cost per Genome
Sep-01 $5,292.39 $95,263,072
Mar-02 $3,898.64 $70,175,437
Sep-02 $3,413.80 $61,448,422
Mar-03 $2,986.20 $53,751,684
Oct-03 $2,230.98 $40,157,554
Jan-04 $1,598.91 $28,780,376
Apr-04 $1,135.70 $20,442,576
Jul-04 $1,107.46 $19,934,346
Oct-04 $1,028.85 $18,519,312
Jan-05 $974.16 $17,534,970
Apr-05 $897.76 $16,159,699
Jul-05 $898.90 $16,180,224
Oct-05 $766.73 $13,801,124
Jan-06 $699.20 $12,585,659
Apr-06 $651.81 $11,732,535
Jul-06 $636.41 $11,455,315
Oct-06 $581.92 $10,474,556
Jan-07 $522.71 $9,408,739
Apr-07 $502.61 $9,047,003
Jul-07 $495.96 $8,927,342
Oct-07 $397.09 $7,147,571
Jan-08 $102.13 $3,063,820
Apr-08 $15.03 $1,352,982
Date Cost per Mb Cost per Genome
Oct-08 $3.81 $342,502
Jan-09 $2.59 $232,735
Apr-09 $1.72 $154,714
Jul-09 $1.20 $108,065
Oct-09 $0.78 $70,333
Jan-10 $0.52 $46,774
Apr-10 $0.35 $31,512
Jul-10 $0.35 $31,125
Oct-10 $0.32 $29,092
Jan-11 $0.23 $20,963
Apr-11 $0.19 $16,712
Jul-11 $0.12 $10,497
Oct-11 $0.09 $7,743
Jan-12 $0.09 $7,666
Apr-12 $0.07 $5,901
Jul-12 $0.07 $5,985
Oct-12 $0.07 $6,618
Jan-13 $0.06 $5,671
Apr-13 $0.06 $5,826
Jul-13 $0.06 $5,550
Oct-13 $0.06 $5,096
Jan-14 $0.04 $4,008
Apr-14 $0.05 $4,920

10. Sequencing MethodSSequencing MethodS
• To determine the order of the nucleotide
bases adenine, guanine, cytosine, and
thymine in a molecule of DNA two methods
were used
1. Maxam and Gilbert; Chemical Sequencing
2. Sanger; Chain Termination Sequencing
• These two are conventional methods
• Robotics and automated sequencing are
based on these methods

11. Maxam and Gilbert MethodMaxam 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-32
P ATP)
II. Purification of the DNA fragment to be sequenced
III. Chemical treatment generates breaks in DNA
IV. Run on the gel

12. Chemical Modification and CleavageChemical Modification and Cleavage
• Ploy nucleotide Kinase radioactive label at one
5' end of the DNA using gamma-32
P
5′ G A C G T G C A A C G A A 3′
32
P 5′ G A C G T G C A A C G A A 3′

13. Chemical Modification and CleavageChemical 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

14. Chemical Modification and CleavageChemical Modification and Cleavage
• Base modification using Hydrazine
– Pyrimidine
• Cytocine
• Thymidine
– Hydrazine----- C+T
– Hydrazine + NaCl--------C
• Cleavage of Sugar Phosphate backbone using
Piperidine

15. DMS
G
G
G
G
FA
G
A
G
G
A
G
A
A
H
C
T
T
C
T
C
C
T
H+S
C
C
C
C
Maxam Gilbert SequencingMaxam Gilbert Sequencing
32
P 5′ G A C G T G C A A C G A 3′

16. Sequencing gels are read from bottom to top (5 to 3 ).′ ′
G G+A T+C C
3′
A
G
C
A
A
C
G
T
G
C
A
G
5′
Longer fragments
Shortest fragments
G
A
Maxam-Gilbert SequencingMaxam-Gilbert Sequencing
32
P 5′ G A C G T G C A A C G A 3′

17. Maxam Gilbert Sequencing: Process SummarizedMaxam Gilbert Sequencing: Process Summarized
1. Label 5’- end of DNA
2. Aliqot DNA sample in 4 tubes
3. Perform base modification reaction
4. Perform Cleavage reaction
5. Perform Gel Electrophoresis
6. Perform Autoradiography
7. Interpret results

18. Sanger; Chain Termination Sequencing
• It is PCR based method
• A modified DNA replication reaction
• Growing chains are terminated by
dideoxynucleotides

19. The 3 -OH group necessary for formation of the phosphodiester bond is missing in ddNTPs′

20. ddATP + ddA
four dNTPs dAdGdCdTdGdCdCdCdG
ddCTP + dAdGddC
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdCddC
ddGTP + dAddG
four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdCddG
ddTTP + dAdGdCddT
four dNTPs dAdGdCdTdGdCdCdCdG
A
C
G
T
Sanger; Chain Termination SequencingSanger; Chain Termination Sequencing
A G C T G C C C G

21. Sequencing gels are read from bottom to top (5 to 3 )′ ′
G A T C
3′
G
G
T
A
A
A
T
C
A
T
G
5′
Longer fragments
Shorter fragments
ddG
ddG
Chain Termination SequencingChain Termination Sequencing

22. Sanger Sequencing: An Example
5’-TACACGATCGA-3’
3’-ATGTGCTAGCT-5’
Denature the sequence
Use only forward primer i.e. using 3’-5

23. 3’-ATGTGCTAGCT-5’
5’-T-3’
5’-TACACGAT-3’
Amplification in ddTTPAmplification in ddTTP
3’-ATGTGCTAGCT-5’
5’-TA-3’
5’-TACA-3’
5’-TACACGA-3’
5’-TACACGATCGA-3’
Amplification in ddATPAmplification in ddATP
Amplification in dGTTPAmplification in dGTTP Amplification in ddCTPAmplification in ddCTP
3’-ATGTGCTAGCT-5’
5’-TACACG-3’
5’-TACACGATCG-3’
3’-ATGTGCTAGCT-5’
5’-TAC-3’
5’-TACAC-3’
5’-TACACGATC-3’

24. Reading Sequence
BAND ddATP ddTTP ddGTP ddCTP
12 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’

25. Sanger Sequencing: Process SummarizedSanger Sequencing: Process Summarized
1. Get enough quantity of DNA (Run PCR)
2. Aliqot DNA into four different tubes
3. Prepare PCR reaction mix as below:
• Primer, taq PM, template(ss DNA), dNTPS (All)
and ddNTPs(ddATP, ddGTP,ddCTP & ddTTP
respectively)
1. Run PCR
2. Perform Gel Electrophoresis
3. Interpret results

26. Thank You
Keep Learning
Thank You
Keep Learning