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DNA Sequencing

LECTURE 6 :. DNA Sequencing . Biotechnology; 3 Credit hours Atta- ur - Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST). Introduction . In 1953, two researchers, namely James Watson and Francis Crick, discovered the basic structure of DNA

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DNA Sequencing

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  1. LECTURE 6: DNA Sequencing Biotechnology; 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST)

  2. Introduction • In 1953, two researchers, namely James Watson and Francis Crick, discovered the basic structure of DNA • DNA is basically a long molecule that stores coded instructions for the cell • The DNA provides a basic blueprint that is responsible for the creation and functioning of cells

  3. Introduction • The information contained in it dictates which cells should grow and when a particular cell should die and how cells should be structured into creating various body parts • For example, the DNA is responsible for determining the quality of our hair, the color and the abundance, or the lack of it

  4. DNA and Nucleotides • DNA stands for Deoxyribonucleic acid • It is present in almost all organisms and it stores long term information that is used to construct an organic body • DNA comprises of a long molecule analogous to a chain, while the links of the chain are called Nucleotides • There are four different nucleotides in the DNA, namely adenine, guanine, cytosine and thymine. They are also called as “A”, “G”, “C” and “T”

  5. The Need for DNA Sequencing • Forensic biology uses DNA sequences to identify the organism which it is unique to • To isolate the genes responsible for genetic diseases like Cystic Fibrosis, Alzheimer’s disease, myotonic dystrophy, etc., which are caused by the inability of genes to work properly • Agriculture has been helped immensely by DNA sequencing • It has allowed scientists to make plants more resistant to insects and pests, by understanding their genes

  6. Genome Sequencing • Genome sequencing is determining the exact order of DNA nucleotides in the genome • In human genome 3.08 billion nucleotides are present, 99% accurately sequenced • DNA sequencing includes several methods and technologies • DNA sequencing can be applied in numerous fields such as diagnostic, biotechnology and biological systems • The first DNA sequences were obtained in the early 1970s

  7. 1870 Miescher: Discovers DNA Avery: Proposes DNA as ‘Genetic Material’ 1940 Watson & Crick: Double Helix Structure of DNA 1953 Holley: Sequences Yeast tRNAAla 1965 Wu: Sequences  Cohesive End DNA 1970 Sanger: Dideoxy Chain Termination Gilbert: Chemical Degradation 1977 1980 Messing: M13 Cloning Hood et al.: Partial Automation 1986 1990 • Cycle Sequencing • Improved Sequencing Enzymes • Improved Fluorescent Detection Schemes 2002 • Next Generation Sequencing • Improved enzymes and chemistry • Improved image processing

  8. Cost of DNA Sequencing

  9. Cost of DNA Sequencing

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

  11. 1- 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 • Chemical Modification of DNA; radioactive labeling at one 5' end of the DNA (typically by a kinase reaction using gamma-32P ATP) • Purification of the DNA fragment to be sequenced • Chemical treatment generates breaks in DNA • Run on the gel

  12. 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 A3′ 32P 5′ G A C G T G C A A C G A A3′

  13. Chemical Modification and Cleavage • Dimethyl sulphate • Purine • Adenine • Guanine • Only DMS------- G • DMS+ Formic acid-------G+A • Piperidine

  14. G A MaxamGilbert Sequencing DMS FA H H+S G G C C A T C G G T C G G C C A T G C C A T 32P 5′ G A C G T G C A A C G A 3′

  15. Chemical Modification and Cleavage • Hydrazine • Pyrimidine • Cytocine • Thymidine • Hydrazine----- C+T • Hydrazine + NaCl--------C

  16. G A MaxamGilbert Sequencing DMS FA H H+S G G C C A T C G G T C G G C C A T G C C A T 32P 5′ G A C G T G C A A C G A 3′

  17. 3′ A G C A A C G T G C A G 5′ G G+A T+C C Longer fragments Shortest fragments G A Maxam-Gilbert Sequencing Sequencing gels are read from bottom to top (5′ to 3′). 32P 5′ G A C G T G C A A C G A 3′

  18. 2- Sanger; Chain Termination Sequencing • Its 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 Sanger; Chain Termination Sequencing A G C T G C CC G A C G T

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

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