dnd sequencing
TRANSCRIPT
TopicDNA
SEQUENCINGChemical Modification Method
Chain Termination Method.
Submitted Dr. Raifullah
Name Amjad KhanReg # 5149Date: 09th December, 2016
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.
• And to analyze gene structure and its relation to gene expression as well as protein conformation
The Need for DNA Sequencing
• Gene isolation• Forensics• Gene Protein Interaction• Cloning• Detecting mutations• Typing microorganisms
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
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
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
Cont…..
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’
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
End