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Polymerase chain reaction medical school

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Polymerase chain reaction medical school

  1. 1. ©2001 Timothy G. Standish V.S.RAVIKIRAN, MSc. Polymerase Chain Reaction
  2. 2. V.S.RAVIKIRAN, MSc., Department of Biochemistry, ASRAM Medical college, Eluru-534005.AP, India. vsravikiran2013@gmail.com
  3. 3. ©2001 Timothy G. Standish V.S.RAVIKIRAN, MSc., Department of Biochemistry, ASRAM Medical college, Eluru-534005.AP, India. vsravikiran2013@gmail.com
  4. 4. ©2001 Timothy G. Standish Polymerase ChainPolymerase Chain ReactionReaction V.S.RAVIKIRAN
  5. 5. ©2001 Timothy G. Standish HistoryHistory The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention A special DNA polymerase (Taq) is used to make many copies of a short length of DNA (100-10,000 bp) defined by primers Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry
  6. 6. ©2001 Timothy G. Standish What PCR Can DoWhat PCR Can Do PCR can be used to make many copies of any DNA that is supplied as a template Starting with one original copy an almost infinite number of copies can be made using PCR “Amplified” fragments of DNA can be sequenced, cloned, probed or sized using electrophoresis
  7. 7. ©2001 Timothy G. Standish What PCR Can DoWhat PCR Can Do Defective genes can be amplified to diagnose any number of illnesses Genes from pathogens can be amplified to identify them (ie. HIV) Amplified fragments can act as genetic fingerprints
  8. 8. ©2001 Timothy G. Standish How PCR WorksHow PCR Works PCR is an artificial way of doing DNA replication Instead of replicating all the DNA present, only a small segment is replicated, but this small segment is replicated many times
  9. 9. ©2001 Timothy G. Standish How PCR WorksHow PCR Works As in replication, PCR involves: –Melting DNA –Priming –Polymerization
  10. 10. ©2001 Timothy G. Standish Initiation - Forming theInitiation - Forming the Replication EyeReplication Eye 3’ 5’ 3’5’ 5’ 5’ 3’ 3’ Origin of Replication 5’ 3’ 3’ 5’ 5’ 3’ 5’ 5’ 5’ 3’ 3’ 3’
  11. 11. ©2001 Timothy G. Standish Leading Strand Laging Strand 3’ 5’ 3’ 5’ Extension - The Replication ForkExtension - The Replication Fork 5’ 5’ 5’ 3’ 3’ 5’3’ 3’ 5’ Single strand binding proteins DNA Polymerase Okazaki fragment RNA Primers Primase 5’ 3’ 5’ Helicase
  12. 12. ©2001 Timothy G. Standish Functions And TheirFunctions And Their Associated EnzymesAssociated Enzymes LigaseJoining nicks DNA PolymerasePolymerizing DNA PrimaseProviding primer EnzymeFunction Helicase SSB Proteins Topisomerase Melting DNA
  13. 13. ©2001 Timothy G. Standish Components of a PCRComponents of a PCR ReactionReaction Buffer (containing Mg++ ) Template DNA 2 Primers that flank the fragment of DNA to be amplified dNTPs Taq DNA Polymerase (or another thermally stable DNA polymerase)
  14. 14. ©2001 Timothy G. Standish PCRPCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature 100 0 50 T i m e 30x 5’3’ 3’5’ 3’5’ 5’ 5’3’ 5’ 3’5’ 5’ 5’ 5’ 5’3’ 3’5’ 3’5’ 5’3’ 5’3’ 5’
  15. 15. ©2001 Timothy G. Standish PCRPCRMelting 94 o C Temperature 100 0 50 T i m e 5’3’ 3’5’
  16. 16. ©2001 Timothy G. Standish PCRPCRMelting 94 o C Temperature 100 0 50 T i m e 3’5’ 5’3’ Heat
  17. 17. ©2001 Timothy G. Standish PCRPCRMelting 94 o C Annealing Primers 50 o C Extension 72 o CTemperature 100 0 50 T i m e 3’5’ 5’3’ 5’ 5’ Melting 94 o C
  18. 18. ©2001 Timothy G. Standish PCRPCRMelting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o CTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ Heat Heat 5’ 5’ 5’
  19. 19. ©2001 Timothy G. Standish PCRPCRMelting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o CTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’
  20. 20. ©2001 Timothy G. Standish PCRPCRMelting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o CTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ Heat Heat
  21. 21. ©2001 Timothy G. Standish PCRPCRMelting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o CTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’
  22. 22. ©2001 Timothy G. Standish Fragments of defined length PCRPCRMelting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o CTemperature 100 0 50 T i m e 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’
  23. 23. ©2001 Timothy G. Standish DNA Between The Primers DoublesDNA Between The Primers Doubles With Each Thermal CycleWith Each Thermal Cycle 0 Cycles Number 1 3 8 2 4 1 2 4 16 5 32 6 64
  24. 24. ©2001 Timothy G. Standish More Cycles = More DNAMore Cycles = More DNA Number of cycles 0 10 15 20 25 30 Size Marker
  25. 25. ©2001 Timothy G. Standish Theoretical Yield Of PCRTheoretical Yield Of PCR Theoretical yield = 2n x y Where y = the starting number of copies and n = the number of thermal cycles = 107,374,182,400 If you start with 100 copies, how many copies are made in 30 cycles? 2n x y = 230 x 100 = 1,073,741,824 x 100
  26. 26. ©2001 Timothy G. Standish How The Functions Of ReplicationHow The Functions Of Replication Are Achieved During PCRAre Achieved During PCR N/A as fragments are short Joining nicks Taq DNA PolymerasePolymerizing DNA Primers are added to the reaction mix Providing primer PCRFunction HeatMelting DNA
  27. 27. ©2001 Timothy G. Standish 27 Type of Gene CyclerType of Gene Cycler Multi Block PCRStandard PCR Gradient PCR Real-Time PCR Gene cyclers available from many suppliers
  28. 28. ©2001 Timothy G. Standish 05/30/15 28 Multiplex PCRMultiplex PCR PCR reactions can be devised in which several targets are amplified simultaneously — often used in diagnostic applications.
  29. 29. ©2001 Timothy G. Standish 05/30/15 29 What is RT-PCRWhat is RT-PCR RT-PCR is Reverse transcription PCR The source of material is mRNA First step is cDNA synthesis by reverse transcriptase at 42ºC Second step is Standard PCR procedure Result is cDNA of gene target Reverse-trascriptionPCRReverse-trascriptionPCR
  30. 30. ©2001 Timothy G. Standish 05/30/15 30 PCRPCR 5’ 3’ 5’3’ 5’ 3’ 5’3’ 5’ 3’ 5’3’ 5’ 3’ 5’3’ 5’ 3’ 5’3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’3’ 5’ 3’5’3’ denaturation (94 o C) primer annealing (50-70 o C) primer extension (72 o C) Next round….. 3’ RT-PCRRT-PCR 5’ AAAAAn TTTTTn 5’ 3AAAAAn TTTTTn first-strand cDNA synthesis by RT 53’ TTTTTn 53’ 5’ TTTTTn 5’ AAAAAn RNase treatment; primer annealing (50-70 o C) primer extension (72 o C) mRNA TTTTTn3’ 5’ 5’ AAAAAn TTTTTn AAAAAn Next round….. An example was described in the article by Halpin et al. (1998). Reverse-trascriptionPCRReverse-trascriptionPCR Basic Reaction of PCR & RT-PCRBasic Reaction of PCR & RT-PCR
  31. 31. ©2001 Timothy G. Standish 05/30/15 31 Applications of PCRApplications of PCR Mutation testing, e.g. cystic fibrosis. Diagnosis or screening of acquired diseases, e.g. AIDS. Genetic profiling in forensic, legal and bio- diversity applications. Site-directed mutagenesis of genes. Quantitation of mRNA in cells or tissues.
  32. 32. THE END THANKS FORYOUR ATTENTION

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