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DNA sequencing
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.
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DNA sequencing
- 2. Topic DNA SEQUENCING Chemical Modification Method ChainTermination Method .
- 3. Submitted Dr. Raifullah NameAmjad Khan Reg # 5149 Date: 09th December, 2016
- 4. ObjectivesObjectives • What isDNA 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 forDNA 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 GilbertMethodMaxam 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 andCleavageChemical 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 andCleavageChemical 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 andCleavageChemical 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 SequencingMaxamGilbert Sequencing 32 P 5′ G A C G T G C A A C G A 3′
- 15. Sequencing gels areread 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 TerminationSequencing • It is PCR based method • A modified DNA replication reaction • Growing chains are terminated by dideoxynucleotides
- 18. ddATP + ddA fourdNTPs 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 areread 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: AnExample 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’ Amplificationin 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’
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- 23. Sanger Sequencing: ProcessSummarizedSanger 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
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