This document provides information about DNA sequencing methods. It discusses that DNA sequencing determines the precise order of nucleotides in DNA. The two main conventional methods described are the Maxam-Gilbert chemical degradation method and the Sanger chain termination method. The Maxam-Gilbert method uses chemical modification and cleavage of DNA bases. The Sanger method is a PCR-based method that uses dideoxynucleotides to terminate DNA chain elongation. Both methods produce fragmented DNA of different lengths that can be resolved on a gel for sequence determination.

2. Topic
DNA
SEQUENCING
Chemical Modification Method
Chain Termination Method
.

3. Submitted Dr. Raifullah
Name Amjad Khan
Reg # 5149
Date: 09th
December, 2016

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

8. 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

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

14. 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′

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

21. 3’-ATGTGCTAGCT-5’
5’-T-3’
5’-TACACGAT-3’
Amplification in
ddTTP
Amplification in
ddTTP
3’-ATGTGCTAGCT-5’
5’-TA-3’
5’-TACA-3’
5’-TACACGA-3’
5’-TACACGATCGA-3’
Amplification in
ddATP
Amplification in
ddATP
Amplification in
dGTTP
Amplification in
dGTTP
Amplification in
ddCTP
Amplification in
ddCTP
3’-ATGTGCTAGCT-5’
5’-TACACG-3’
5’-TACACGATCG-3’
3’-ATGTGCTAGCT-5’
5’-TAC-3’
5’-TACAC-3’
5’-TACACGATC-3’

22. Reading the sequencings

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

24. Thank You
End