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  1. 1. Gene amplification through PCR Presented By P.UMA DEVI RVM 08-26 MVSC 1 ND YEAR
  2. 2. Contents <ul><li>PCR Definition </li></ul><ul><li>Components of the reaction mixture </li></ul><ul><li>PCR primer design guidelines </li></ul><ul><li>Steps in PCR </li></ul><ul><li>Variations on the basic PCR technique </li></ul><ul><li>Comparison of PCR and Gene cloning </li></ul><ul><li>Applications of PCR </li></ul><ul><li>Problems related to PCR </li></ul>
  3. 3. What is PCR? <ul><li>P olymerase C hain R eaction is an in vitro technique for the amplification of a specific sequence of DNA Which is used for further testing. </li></ul>
  4. 4. <ul><li>Kary Mullis (1987) </li></ul><ul><li>Cetus Corporation (A Biotech Company of United States) </li></ul><ul><li>Nobel Prize 1993 </li></ul>
  5. 5. Components of the reaction mixture <ul><li>Template DNA. </li></ul><ul><li>Primers (forward and reverse) </li></ul><ul><li>dNTPs </li></ul><ul><li>Taq DNA Polymerase </li></ul><ul><li>Buffer solution </li></ul><ul><li>Divalent cations </li></ul><ul><li>Sterile deionized water </li></ul>
  6. 6. Template DNA <ul><li>It contains the DNA region to be amplified </li></ul><ul><li>Range - 1-2 µl ( for a total reaction mixture of 10 µl) </li></ul>
  7. 7. Primers <ul><li>Short Single stranded oligonucleotides </li></ul><ul><li>They are complementary to the 5' or 3' ends of the DNA region </li></ul><ul><li>Range - 1 µl ( for a total reaction mixture of 10 µl) </li></ul>TT AA C GG CC TT AA . . . TTT AAA CC GG TT AA TT G CC GG AA TT . . . . . . . . . .> and <. . . . . . . . . . AAA TTT GG CC AA TT AA C GG CC TT AA . . . TTT AAA CC GG TT
  8. 8. PCR Primer Design Guidelines <ul><li>Primer Length: </li></ul><ul><li>Optimal length of PCR primers is 18-22 bp </li></ul><ul><li>TT AA C GG CC TT AA ….. TTT AAA CC GG TT </li></ul><ul><li>AA TT G CC GG AA TT ........> </li></ul>
  9. 9. <ul><li>Primer Melting Temperature: (Tm) </li></ul><ul><li>Temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. </li></ul><ul><li>Range - 52-58 ° C </li></ul><ul><li>Formula </li></ul><ul><li>Tm = 4 (G+C) + 2 (A+T) </li></ul><ul><li>( GCAT no. of respective nucleotides in the primer) </li></ul>
  10. 10. <ul><li>GC Content 40-60%. </li></ul><ul><li>GC Clamp </li></ul><ul><li>Presence of G or C bases within the last five bases from the 3' end of primers </li></ul><ul><li>Promotes specific binding at the 3' end due to the stronger bonding of G and C bases </li></ul>
  11. 11. <ul><li>Primer Secondary Structures : produced by intermolecular or intramolecular interactions </li></ul><ul><li>Lead to poor or no yield of the product. </li></ul><ul><li>Ex - Hairpins </li></ul><ul><li>Dimers </li></ul>
  12. 12. <ul><li>Hairpins Intramolecular interaction within the primer </li></ul>
  13. 13. <ul><li>Self Dimer </li></ul><ul><li>They formed by intermolecular interactions between the two primers, where the primer is homologous to itself. </li></ul><ul><li>They reduce the product yield. </li></ul>
  14. 14. <ul><li>Repeats A di-nucleotide occurring many times consecutively. </li></ul><ul><li>They should be avoided because they can misprime. </li></ul><ul><li>Ex. A T A T A T A T A T A T ……….. </li></ul><ul><li>Acceptable di-nucleotide repeats are maximum 4 </li></ul>
  15. 15. <ul><li>Runs </li></ul><ul><li>Primers with long runs of a single base . </li></ul><ul><li>Ex . A GCGGGGG A T GGGG ……….. </li></ul><ul><li>The maximum number of runs accepted are 4 </li></ul>
  16. 16. <ul><li>Avoid Cross homology Primers designed for a sequence must not amplify other genes in the mixture. </li></ul><ul><li>Position Sequence close to the 3‘ end preferred most frequently. </li></ul>
  17. 17. dNTPs <ul><li>De oxy nucleotide triphosphate (dATP, dGTP, dTTP, dCTP) </li></ul><ul><li>They are the building blocks from which the DNA polymerases synthesizes a new DNA strand. </li></ul><ul><li>Range - 0.5 µl (for 10µl reaction mixture) </li></ul>
  18. 18. dNTPs in the reaction mix
  19. 19. Taq DNA Polymerase <ul><li>T hermus aq aticus </li></ul><ul><li>Range 0.2ul of (in 10µl of reaction mix) </li></ul><ul><li>It assebles a new DNA strand from dNTPs </li></ul>
  20. 20. <ul><li>Buffer solution </li></ul><ul><li>Contains Divalent cations like Mg+2 </li></ul><ul><li>Provides suitable chemical environment for optimum activity and stability of the DNA polymerase </li></ul><ul><li>Range - 1 µl ( for a total reaction mixture of 10 µl) </li></ul><ul><li>Sterile deionized water </li></ul><ul><li>It’s quantity is variable </li></ul>
  21. 21. Steps in PCR <ul><li>Initialization </li></ul><ul><li>Denaturation </li></ul><ul><li>Annealing </li></ul><ul><li>Extension / Elongation </li></ul><ul><li>Final elongation </li></ul><ul><li>Final hold </li></ul><ul><li>Initialization step </li></ul><ul><li>Heating the reaction to a temperature of </li></ul><ul><li>94-96°C for 1-9 minutes. </li></ul>
  22. 22. <ul><li>Denaturation step </li></ul><ul><li>94-98°C for 20-30 seconds . </li></ul><ul><li>Denaturation of DNA template by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA . </li></ul>
  23. 25. <ul><li>Annealing step </li></ul><ul><li>50-65°C for 20-40 seconds </li></ul><ul><li>Stable DNA-DNA hydrogen bonds are formed </li></ul><ul><li>The polymerase binds to the primer-template hybrid and begins DNA synthesis. </li></ul>
  24. 27. Extension/elongation step <ul><li>75-80°C </li></ul><ul><li>At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template by adding dNTPs in 5' to 3' direction. </li></ul>
  25. 28. <ul><li>Final elongation </li></ul><ul><li>70-74°C for 5-15 minutes </li></ul><ul><li>To ensure that any remaining single-stranded DNA is fully extended. </li></ul><ul><li>Final hold </li></ul><ul><li>4-15°C for an indefinite time </li></ul><ul><li>short-term storage of the reaction </li></ul>
  26. 31. Allele- Specific PCR <ul><li>Selective PCR amplification of the alleles to detect single nucleotide polymorphism (SNP) </li></ul><ul><li>Selective amplification is usually achieved by designing a primer such that the primer will match or mismatch one of the alleles at the 3’ end of the primer. </li></ul>
  27. 32. Asymmetric PCR <ul><li>It is used for DNA sequencing </li></ul><ul><li>The two primers are used in the 100:1 ratio so that after 20-25 cycles of amplification one primer is exhausted thus single stranded DNA is produced in the next 5-10 cycles </li></ul>
  28. 33. Real Time PCR <ul><li>Quantitative real time PCR (Q-RT PCR) </li></ul><ul><li>It is used to amplify and simultaneously quantify a target target DNA molecule </li></ul><ul><li>Real time PCR using DNA dyes </li></ul><ul><li>Fluorescent reporter probe method </li></ul>
  29. 34. Real Time PCR
  30. 35. Helicase-dependent amplification <ul><li> Constant temperature is used rather than cycling through denaturation and annealing/extension cycles. </li></ul><ul><li> DNA Helicase , an enzyme that unwinds DNA, is used in place of thermal denaturation. </li></ul>
  31. 36. Intersequence-specific PCR (ISSR): <ul><li>A PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths. </li></ul>
  32. 37. Inverse PCR <ul><li> A method used to allow PCR when only one internal sequence is known. </li></ul><ul><li> This is especially useful in identifying flanking sequences of various genomic inserts. </li></ul>
  33. 39. Anchored PCR <ul><li>When sequence of only one end of the desired segment of gene is known,the primer complimentary to the 3' strand of this end is used to produce several copies of only one strand of the gene. </li></ul>
  34. 41. RT-PCR ( Reverse Transcription PCR) <ul><li> It is used to amplify, isolate or identify a known sequence from a cellular or tissue RNA . </li></ul><ul><li> RT-PCR is widely used in expression profiling , to determine the expression of a gene or to identify the sequence of an RNA transcript. </li></ul><ul><li>RACE-PCR </li></ul><ul><li> Used to obtain 3' and 5' end sequence of cDNA transcripts </li></ul>
  35. 43. Comparison PCR - Polymerase Chain Reaction and Gene Cloning Two to four days Four hours Time for a typical experiment 12. Required Not required User’s skill 11. More Less Cost 10. Less More Applications 9. More Less Error probability 8. Yes No Labour intensive 7. No Yes Automation 6. Restriction enzymes, Ligase, vector. bacteria DNA polymerase (Taq polymerase) Biological reagents required 5. Microgram (m) Nanogram (ng) Quantity of starting material 4. Last step First step Selectivity of the specific segment from complex DNA 3. In vitro and in vivo In vitro Manipulation 2. Selective amplification of specific sequence Selective amplification of specific sequence Final result 1. Gene cloning PCR   Parameter
  36. 44. Application of PCR <ul><li>Cloning a Gene encoding a known protein </li></ul><ul><li>Amplification of old DNA </li></ul><ul><li>Amplifying cloned DNA from Vectors </li></ul><ul><li>Rapid Amplification of cDNA ends </li></ul><ul><li>Detecting Bacterial or Viral Infection </li></ul><ul><li>● AIDS infection </li></ul><ul><li>● Tuberculosis (Mycobacterium tuberculosis) </li></ul>
  37. 45. <ul><li>Genetics Diagnosis </li></ul><ul><li>Diagnosing inherited disorders </li></ul><ul><li>Cystic fibrosis </li></ul><ul><li>Muscular dystrophy </li></ul><ul><li>Haemophilia A and B </li></ul><ul><li>Sickle cell anaemia </li></ul><ul><li>Diagnosing cancer </li></ul><ul><li>Blood group typing. </li></ul>
  38. 46. Problems with PCR <ul><li>Polymerase errors </li></ul><ul><li>Polymerase lacks exonuclease activity </li></ul><ul><li>Size limitations </li></ul><ul><li>PCR works readily with DNA of lengths two to three thousand basepairs </li></ul><ul><li>Non specific priming </li></ul>
  39. 47. THANK YOU