4. ObjectivesObjectives
• What is DNA Sequencing ?
• History of development
• Basic Methods- Chain
termination and Chemical
modification method
5. 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.
• And to analyze gene structure and its relation to gene
expression as well as protein conformation
6. The Need for DNA SequencingThe Need for DNA Sequencing
• Gene isolation
• Forensics
• Gene Protein Interaction
• Cloning
• Detecting mutations
• Typing microorganisms
7. 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
9. 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
10. 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
11. 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′
12. 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
13. 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. 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′
16. 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
17. Sanger; Chain Termination Sequencing
• It is PCR based method
• A modified DNA replication reaction
• Growing chains are terminated by
dideoxynucleotides
18. 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
Sequencing
Sanger; Chain Termination
Sequencing
A G C T G C C C G
19. 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
Cont…..Cont…..
20. Sanger Sequencing: An Example
5’-TACACGATCGA-3’
3’-ATGTGCTAGCT-5’
Denature the sequence
Use only forward primer i.e. using 3’-5
23. 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
