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PCR types and applications

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Basics of PCR principle, types and applications

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PCR types and applications

  1. 1. Polymerase Chain ReactionPolymerase Chain Reaction (PCR) and Its(PCR) and Its ApplicationsApplications S.Karthikumar.,M.Sc.,M.Phil.,M.Tech.,(Ph.D) Assistant Professor, Department of Biotechnology Kamaraj College of Engineering and Technology Virudhunagar-626001, Tamilnadu, INDIA karthikumarbt@kcetvnr.org 1
  2. 2. What is PCR?What is PCR? PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. It was invented in 1983 by Dr. Karry Mullis, for which he received the Nobel Prize in Chemistry in 1993. karthikumarbt@kcetvnr.org 2
  3. 3. Did He Really Invent PCR? • The basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971: – Kleppe et al. (1971) J. Mol. Biol. 56, 341-346. • Progress was limited by primer synthesis and polymerase purification issues. • Mullis properly exploited amplification. karthikumarbt@kcetvnr.org 3
  4. 4. What is PCR? :What is PCR? : Why “Polymerase”?Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. karthikumarbt@kcetvnr.org 4
  5. 5. What is PCR? :What is PCR? : Why “Chain”?Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on. karthikumarbt@kcetvnr.org 5
  6. 6. What is PCR? :What is PCR? : The “Reaction” ComponentsThe “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme karthikumarbt@kcetvnr.org 6
  7. 7. The Basics of PCR Cycling • 30–35 cycles each comprising: – denaturation (95°C), 30 sec. – annealing (55–60°C), 30 sec. – extension (72°C), time depends on product size. karthikumarbt@kcetvnr.org 7
  8. 8. The ReactionThe Reaction THERMOCYCLERPCR tube karthikumarbt@kcetvnr.org 8
  9. 9. Taq polymerase Thermus aquaticus, a thermophilic bacteria discovered in 1969 in hot spring of Yellowstone National park . It can tolerate high temperature. The DNA polymerase (Taq polymerase) was isolated. karthikumarbt@kcetvnr.org 9
  10. 10. Thermostable Polymerases Polymerase T ½, 95o C Extension Rate (nt/sec) Type of ends Source Taq pol 40 min 75 3’A T. aquaticus Amplitaq (Stoffel fragment) 80 min >50 3’A T. aquaticus Vent* 400 min >80 95% blunt Thermococcus litoralis Deep Vent* 1380 min ? 95% blunt Pyrococcus GB-D Pfu >120 min 60 Blunt Pyrococcus furiosus Tth* (RT activity) 20 min >33 3’A T. thermophilus *Have proof-reading functions and can generate products over 30 kbp karthikumarbt@kcetvnr.org 10
  11. 11. • Taq: Thermus aquaticus (most commonly used) – Sequenase: T. aquaticus YT-1 – Restorase (Taq + repair enzyme) • Tfl: T. flavus • Tth: T. thermophilus HB-8 • Tli: Thermococcus litoralis • Carboysothermus hydrenoformans (RT-PCR) • P. kodakaraensis (Thermococcus) (rapid synthesis) • Pfu: Pyrococcus furiosus (fidelity) – Fused to DNA binding protein for processivity karthikumarbt@kcetvnr.org 11
  12. 12. A Standard PCR Reaction Mix 0.25 mM each primer 0.2 mM each dATP, dCTP, dGTP, dTTP 50 mM KCl 10 mM Tris, pH 8.4 1.5 mM MgCl2 2.5 units polymerase 102 - 105 copies of template 50 ml reaction volume karthikumarbt@kcetvnr.org 12
  13. 13. Denature (heat to 95o C) Lower temperature to 56o C Anneal with primers Increase temperature to 72o C DNA polymerase + dNTPs karthikumarbt@kcetvnr.org 13
  14. 14. karthikumarbt@kcetvnr.org 14
  15. 15. karthikumarbt@kcetvnr.org 15
  16. 16. PCR Products on Agarose gel karthikumarbt@kcetvnr.org 16
  17. 17. Combinations Of Cycle Temperatures TEMP FOR COMMENTS 94-60-72 Perfect, long primers Higher temp can be used; maximum annealling temp 94-55-72 Good or perfectly matched primers between 19-24 nt Standard conditions 94-50-72 Adequate primers Allows 1-3 mismatches/20 nt 94-48-68 Poorly matched primers Allows 4-5 mismatches/20 nt 94-45-65 Unknown match, likely poor Primers of questionable quality, long-shot PCR 94-37-65 Hail Mary Uncontrolled results karthikumarbt@kcetvnr.org 17
  18. 18. Do Errors Matter in PCR? • Yes, if you want to clone the amplified DNA — an individual molecule may harbour several mutations. • Use a proof-reading thermo-stable enzyme rather than Taq. karthikumarbt@kcetvnr.org 18
  19. 19. How Big A Target? • Amplification products are typically in the size range 100-1500 bp. • Longer targets are amplifiable — >25 kb. • Requires modified reaction buffer, cocktails of polymerases, and longer extension times. • Limited by the integrity of the starting target DNA — > 50 kb. karthikumarbt@kcetvnr.org 19
  20. 20. Can I PCR Amplify RNA? • Not directly — the DNA polymerase requires a DNA template and will not copy RNA. • mRNA can first be copied into cDNA using reverse transcriptase. • cDNA is a template for PCR — it need not be double-stranded. karthikumarbt@kcetvnr.org 20
  21. 21. Designing PCR Primers • Primers should be ~20 bases long. • The G/C content should be 45–55%. • The annealing temperatures should be within 1°C of one another. • The 3´-most base should be a G or C. • The primers must not base pair with each other or with themselves or form hairpins. • Primers must avoid repetitive DNA regions. karthikumarbt@kcetvnr.org 21
  22. 22. Primers That Form Hairpins • A primer may be self-complementary and be able to fold into a hairpin: 5´-GTTGACTTGATA ||||| T 3´-GAACTCT • The 3´ end of the primer is base-paired, preventing it annealing to the target DNA. karthikumarbt@kcetvnr.org 22
  23. 23. Primers That Form Dimers • A primer may form a dimer with itself or with the other primer. 5´-ACCGGTAGCCACGAATTCGT-3´ |||||||||| 3´-TGCTTAAGCACCGATGGCCA-5´ • Primer dimers can be an excellent, but unwanted, substrate for the Taq polymerase. karthikumarbt@kcetvnr.org 23
  24. 24. Will Other Genes Amplify Too? • The primers have been designed on the basis of the DNA sequence of a single gene. • Might the primers also amplify other segments whose sequence we have not taken into account? karthikumarbt@kcetvnr.org 24
  25. 25. Type of PCR • Single PCR • Nested PCR • Multiplex PCR • Reverse transcriptase PCR • Asymmetric PCR • Quantitative PCR karthikumarbt@kcetvnr.org 25
  26. 26. Nested PCR karthikumarbt@kcetvnr.org 26
  27. 27. Applications of PCRApplications of PCR Molecular Identification Sequencing Genetic Engineering • DNA fingerprinting • Classification of organisms • Genotyping • Pre-natal diagnosis • Mutation screening • Drug discovery • Genetic matching • Detection of pathogens • Bioinformatics • Genomic cloning • Human Genome Project • Site-directed mutagenesis • Gene expression studies karthikumarbt@kcetvnr.org 27
  28. 28. MMOLECULAROLECULAR IIDENTIFICATIONDENTIFICATION karthikumarbt@kcetvnr.org 28
  29. 29. karthikumarbt@kcetvnr.org 29
  30. 30. karthikumarbt@kcetvnr.org 30
  31. 31. Detection Of PathogensDetection Of Pathogens karthikumarbt@kcetvnr.org 31
  32. 32. SSEQUENCINGEQUENCING Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP) NO polymerisation after a dideoxynucleotide! karthikumarbt@kcetvnr.org 32
  33. 33. karthikumarbt@kcetvnr.org 33
  34. 34. Reading Classical Sequencing GelsReading Classical Sequencing Gels karthikumarbt@kcetvnr.org 34
  35. 35. Enzyme linked oligonucleotide sorbent assay karthikumarbt@kcetvnr.org 35

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