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Dna Sequencing

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Dna Sequencing

  1. 1. <ul><li>PRESENTED BY </li></ul><ul><li>MARIAM RAZI </li></ul><ul><li>BS medical technology 6 th semester </li></ul>DNA SEQUENCING
  2. 2. DNA <ul><li>Deoxyribonucleic acid (DNA) is a nucleic acid that functions include </li></ul><ul><li>Storage of genetic information </li></ul><ul><li>Self-duplication & inheritance. </li></ul><ul><li>Expression of the genetic message . </li></ul><ul><li>DNA’s major function is to code for proteins. </li></ul><ul><li>Information is encoded in the order of the nitrogenous bases. </li></ul>
  3. 3. Watson & Crick Model   <ul><li>DNA is composed of 2 chains of nucleotides that form a double helix shape. </li></ul><ul><li>The two strands are antiparallel. </li></ul><ul><li>The backbone of the DNA molecule is composed of alternating phosphate groups and sugars. </li></ul><ul><li>The complimentary nitrogenous bases form hydrogen bonds between the strands. </li></ul><ul><li>A is complimentary to T and G is complimentary to C. </li></ul>
  4. 4. DNA SEQUENCING <ul><li>Determining the order of bases in a section of DNA </li></ul>
  5. 5. PURPOSE <ul><li>Deciphering “code of life” </li></ul><ul><li>Detecting mutations </li></ul><ul><li>Typing microorganisms </li></ul><ul><li>Identifying human halotypes </li></ul><ul><li>Designating polymorphisms </li></ul>
  6. 6. DNA SEQUENCING METHODS <ul><li>Historically there are two main methods of DNA sequencing: </li></ul><ul><li> 1.Maxam and Gilbert method </li></ul><ul><li>2.Sanger method </li></ul><ul><li>Modern sequencing equipment uses the principles of the Sanger technique. </li></ul>
  7. 7. MAXAM & GILBERT METHOD <ul><li>A. M. Maxam and W.Gilbert-1977 </li></ul><ul><li>The sequence of a double-stranded or single-stranded DNA molecule is determined by treatment with chemicals that cut the molecule at specific nucleotide positions. </li></ul>
  8. 8. PRINCIPLE : <ul><li>Chemical degradation </li></ul><ul><li>Reaction in two stages: </li></ul><ul><li>Chemical modification of the bases </li></ul><ul><li>Modified base is removed from its sugar, pyperidin cleaves phosphodiester bonds 5’ and 3’ and base is released </li></ul>
  9. 11. SANGER METHOD <ul><li>Most common approach used to sequencing DNA. </li></ul><ul><li>Invented by Frederick Sanger - 1977 </li></ul><ul><li>Nobel prize - 1980 </li></ul><ul><li>Also termed as chain termination or dideoxy method </li></ul>
  10. 12. SANGER METHOD TERMED AS CHAIN TERMINATION METHOD <ul><ul><ul><li>This method uses dideoxynucleotide triphosphates </li></ul></ul></ul><ul><ul><ul><li>(ddNTPs) chain terminators : </li></ul></ul></ul><ul><ul><ul><li>which have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs). </li></ul></ul></ul><ul><ul><ul><li>Therefore in a synthesis reaction, if a dideoxynucleotide is </li></ul></ul></ul><ul><ul><ul><li>added instead of the normal deoxynucleotide, the synthesis </li></ul></ul></ul><ul><ul><ul><li>stops at that point because the 3’OH necessary for the </li></ul></ul></ul><ul><ul><ul><li>addition of the next nucleotide is absent. </li></ul></ul></ul>
  11. 13. DEOXY VERSUS DIDEOXY
  12. 14. PRINCIPLE : <ul><li>The sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains. </li></ul><ul><li>These chains terminating at specific nucleotide positions. </li></ul><ul><li>Separate by gel electrophoresis </li></ul><ul><li>Read DNA sequence </li></ul>
  13. 15. REQIREMENTS <ul><li>DNA sequencing is performed in four separate tubes, </li></ul><ul><li>each containing </li></ul><ul><li>         </li></ul><ul><li>i.   Single stranded DNA to be sequenced </li></ul><ul><li>           ii.  DNA polymerase </li></ul><ul><li>           iii.  Primers </li></ul><ul><li>          iv.  The four dNTPs (dATP, dCTP, dTTP and dGTP) </li></ul><ul><li>          v.    Small amount of one of the four ddNTPs </li></ul><ul><li>(ddATP or ddCTP or ddTTP or ddGTP) </li></ul><ul><li>Either the primers or the dNTPs are radiolabeled with 32P </li></ul>
  14. 16. HOW DOES THE DNA TEMPLATE OBTAINED? <ul><li>The DNA can be cloned in a plasmid vector. </li></ul><ul><li>The DNA can be cloned in a bacteriophage M13 vector. </li></ul><ul><li>PCR can be used to generate single-stranded DNA. </li></ul>
  15. 18. COMPARISON Automation is not available Automation Double-stranded or single- stranded DNA Single-stranded DNA. <ul><ul><li>Breaks DNA at different nucleotides </li></ul></ul><ul><ul><li>Termination of chain elongation </li></ul></ul><ul><ul><li>Requires DNA </li></ul></ul>Requires DNA synthesis <ul><ul><li>Chemical </li></ul></ul><ul><ul><li>Enzymatic </li></ul></ul>Maxam Gilbert Method Sanger Method
  16. 19. Next Generation Technologies: <ul><ul><li>Solexa : Based whole genome sequencing </li></ul></ul><ul><ul><li>SOLiD : Ligation and detection </li></ul></ul><ul><ul><li>454 : Pyrosequencing </li></ul></ul><ul><ul><li>Helicos : Single molecule sequencing </li></ul></ul>
  17. 20. THANK YOU

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