Dna sequencing

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Dna sequencing

  1. 1. EVALUATION SEMINAR ON DNA SEQUENCING PRESENTED BY AMIT SHRESTHA M-PHARM 9/25/2013 1MALLIGE COLLEGE OF PHARMACY
  2. 2. DNA Deoxyribonucleic acid (DNA) is a nucleic acid that functions include  Storage of genetic information  Self-duplication & inheritance. Expression of the genetic message. DNA’s major function is to code for proteins. Information is encoded in the order of the nitrogenous bases. Along with RNA and proteins, DNA is one of the three major macromolecules essential for all known forms of life. Most DNA molecules are double-stranded helices, consisting of two long biopolymers of simpler units called nucleotides—each nucleotide is composed of a nucleobase (guanine, adenine, thymine, and cytosine), 9/25/2013 2MALLIGE COLLEGE OF PHARMACY
  3. 3. Watson & Crick Model 9/25/2013 3MALLIGE COLLEGE OF PHARMACY
  4. 4. DNA SEQUENCING Determining the order of bases in a section of DNA four bases—adenine guanine cytosine thymine 9/25/2013 4MALLIGE COLLEGE OF PHARMACY
  5. 5. PURPOSE • Deciphering “code of life” • Detecting mutations • Typing microorganisms • Identifying human halotypes • Designating polymorphisms 9/25/2013 5MALLIGE COLLEGE OF PHARMACY
  6. 6. DNA SEQUENCING METHODS Historically there are two main methods of DNA sequencing: 1.Maxam and Gilbert method 2.Sanger method Modern sequencing equipment uses the principles of the Sanger technique. 9/25/2013 6MALLIGE COLLEGE OF PHARMACY
  7. 7. MAXAM & GILBERT METHOD • A. M. Maxam and W.Gilbert-1977 • 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. 9/25/2013 7MALLIGE COLLEGE OF PHARMACY
  8. 8. PRINCIPLE : Chemical degradation Reaction in two stages: • Chemical modification of the bases • Modified base is removed from its sugar, pyperidin cleaves phosphodiester bonds 5’ and 3’ and base is released 9/25/2013 8MALLIGE COLLEGE OF PHARMACY
  9. 9. The method requires radioactive labelling at one end and purification of the DNA fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each molecule. The fragments in the four reactions are arranged side by side in geL electrophoresis for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabelled DNA fragment, from which the sequence may be inferred. PROCEDURE 9/25/2013 9MALLIGE COLLEGE OF PHARMACY
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  11. 11. SANGER METHOD • Most common approach used to sequencing DNA. • Invented by Frederick Sanger - 1977 • Nobel prize - 1980 • Also termed as chain termination or dideoxy method 9/25/2013 11MALLIGE COLLEGE OF PHARMACY
  12. 12. SANGER METHOD TERMED AS CHAIN TERMINATION METHOD This method uses dideoxynucleotide triphosphates (ddNTPs) chain terminators : which have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs). Therefore in a synthesis reaction, if a dideoxynucleotide is added instead of the normal deoxynucleotide, the synthesis stops at that point because the 3’OH necessary for the addition of the next nucleotide is absent. 9/25/2013 12MALLIGE COLLEGE OF PHARMACY
  13. 13. DEOXY VERSUS DIDEOXY 9/25/2013 13MALLIGE COLLEGE OF PHARMACY
  14. 14. PRINCIPLE : The sequence of a single-stranded DNA molecule is determined by enzymatic synthesis of complementary polynucleotide chains. These chains terminating at specific nucleotide positions. Separate by gel electrophoresis Read DNA sequence 9/25/2013 14MALLIGE COLLEGE OF PHARMACY
  15. 15. REQIREMENTS DNA sequencing is performed in four separate tubes, each containing i. Single stranded DNA to be sequenced ii. DNA polymerase iii. Primers iv. The four dNTPs (dATP, dCTP, dTTP and dGTP) v. Small amount of one of the four ddNTPs (ddATP or ddCTP or ddTTP or ddGTP) Either the primers or the dNTPs are radiolabeled with 32P 9/25/2013 15MALLIGE COLLEGE OF PHARMACY
  16. 16. PROCEDURE In the Sanger method, the DNA strand to be analyzed is used as a template and DNA polymerase is used, in a PCR reaction, to generate complimentary strands using primers. Four different PCR reaction mixtures are prepared, each containing a certain percentage of dideoxynucleoside triphosphate (ddNTP) analogs to one of the four nucleotides (ATP, CTP, GTP or TTP). Synthesis of the new DNA strand continues until one of these analogs is incorporated, at which time the strand is prematurely truncated. Each PCR reaction will end up containing a mixture of different lengths of DNA strands, all ending with the nucleotide that was dideoxy labeled for that reaction. Gel electrophoresis is then used to separate the strands of the four reactions, in four separate lanes, and determine the sequence of the original template based on what lengths of strands end with what nucleotide.9/25/2013 16MALLIGE COLLEGE OF PHARMACY
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  21. 21. COMPARISON Sanger Method Maxam Gilbert Method Enzymatic Chemical Requires DNA synthesis Requires DNA Termination of chain elongation Breaks DNA at different nucleotides Automation Automation is not available Single-stranded DNA. Double-stranded or single- stranded DNA 9/25/2013 21MALLIGE COLLEGE OF PHARMACY
  22. 22. 9/25/2013 MALLIGE COLLEGE OF PHARMACY 22 Applications of DNA Sequencing • Forensics – Identify individuals – Determine the paternity of a child – Identifies endangered and protected species • Medicine – Detect genes that are hereditary or cause diseases • Agriculture – Map the genome of microorganisms
  23. 23. 9/25/2013 MALLIGE COLLEGE OF PHARMACY 23 Future of DNA Sequencing • Projects might focus on researching: – The links to develop lifestyle – Genomic and cardiovascular disease – Early detections of cancer
  24. 24. THANK YOU 9/25/2013 24MALLIGE COLLEGE OF PHARMACY

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