Abbas Morovvati


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Abbas Morovvati

  1. 1. Polymerase Chain Reaction
  2. 2. What is the Polymerase Chain Reaction? DNA dsDNA Amplify4/28/2012 2
  3. 3. What is it?
  4. 4. History One of the most powerful tools in molecular biology Invented by Kary Mullis in 1983, resulting in his Nobel Prize in Chemistry First published account appeared in 1985. Awarded Nobel Prize for Chemistry in 1993.4/28/2012 4
  5. 5. The reaction mixture1. DNA (purified or a crude extract)2. Primers specific for the target DNA3. Free nucleotides (A, G, T, C)4. DNA polymerase5. Buffer (containing magnesium)
  6. 6. The reaction mixture 1- DNA template that contains the DNA region (target) to be amplified. 2- One or more primers, which are complementary to the DNA regions at the 5 (five prime) and 3 (three prime) ends of the DNA region. 3- A DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70 C.4/28/2012 6
  7. 7. The reaction mixture , (dNTPs) from which the DNA polymerase builds the new DNA , which provides a suitable chemical environment for the DNA Polymerase , or ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis Monovalent cation ions.4/28/2012 7
  8. 8. Primers• On the other hand, the length of a primer is limited by: the maximum temperature allowed to be applied in order to melt it, as increases with the of the primer that are too high, i.e., above , can cause problems: since the is at such temperatures• The of a primer is generally from 15 to 40 , with a between calculating: Tm =4(G+C)+2(A+T) Software
  9. 9. Primers• The DNA fragment to be amplified is determined by selecting primers• Primers are : short, artificial DNA strands often not more than 50 and usually only 18 to 25 base pairs long that are complementary to the beginning or the end of the DNA fragment to be amplified• They anneal by adhering to the DNA template at these starting & ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand
  10. 10. Primer 3 terminus• Primer 3 terminus design is critical to PCR success since the primer extends from the 3 end• The 3 end should not be complementary over greater than 3-4 bases to any region of the other primer (or even the same primer) used in the reaction and must provide correct base matching to the template• There are computer programs to help design primers Genrunner
  11. 11. PCR - DNAC G 5’ 3’Target DNA 3’ 5’
  12. 12. reverseforward
  13. 13. The basic protocol1. Denaturation of DNA to single strands2. Annealing of primers to DNA3. Extension by polymerase4. Repeat 30-35 times
  14. 14. ProcedureThe PCR process usually consists of 20 -35each cycle consists of :1. The has to be heated to (or 98 C if extremely thermostable polymerases are used)in order to separate the strandsThis step is called denaturing:it breaks apart the that connect the two DNA strandsPrior to the first cycle:the DNA is often denatured for an to ensure that the and the ,have completely separated and are now usually , but up to minutesAlso certain polymerases at this step (
  15. 15. Procedure2. After separating the DNA strands, the temperature is so :the primers can themselves to thethe temperature of this stage on the and is usually their Tm (45-60 C)A wrong temperature during the annealing step can result in :primers not binding to the template DNA at allor binding at randomTime: 1-2 minutes
  16. 16. Procedure3. Finally, the DNA polymerase has to copy the DNA strands It starts at the annealed primer and works its way along the DNA strand this step is called elongation the elongation temperature depends on the DNA polymerase: Taq polymerase elongates optimally at a temperature of 72º C• The time for this step depends:1. both on the DNA polymerase itself2. and on the length of the DNA fragment to be amplified as a rule-of-thumb, this step takes 1 minute per 1000 bp• A final elongation step is frequently used after the last cycle to ensure that any remaining single stranded DNA is completely copied, this differs from all other elongation steps, only in that it is longer, typically 10-15 minutes
  17. 17. Primers forward 5’ 3’Target DNA 3’ 5’ reverse
  18. 18. The basic protocol--denaturation 5’ 3’Target DNA 3’ 5’ 95oC 5’ 3’ 3’ 5’
  19. 19. The basic protocol--annealing 5’ 3’Target DNA 3’ 5’ A B primers ~55oC 5’ 3’ B 5’ 5’ A 3’ 5’
  20. 20. The basic protocol--extension 5’ 3’Target DNA 3’ 5’ 72oC 5’ 3’ Taq polymerase 5’ 5’ 3’ 5’
  21. 21. The basic protocol--extension 5’ 3’Target DNA 3’ 5’ 72oC 5’ 3’ 5’ 5’ 3’ 5’
  22. 22. The basic protocol--denaturation 5’ 3’Target DNA 3’ 5’ 95oC 5’ 3’ 3’ 5’
  23. 23. The basic protocol--annealing 5’ 3’Target DNA 3’ 5’ A B primers ~55oC 5’ 3’ B 5’ 5’ A 3’ 5’
  24. 24. The basic protocol--extension 5’ 3’Target DNA 3’ 5’ 72oC 5’ 3’ Taq polymerase 5’ 5’ 3’ 5’
  25. 25. The basic protocol--extension 5’ 3’Target DNA 3’ 5’ 72oC 5’ 3’ 5’ 5’ 3’ 5’
  26. 26. One One billion in about 2 hours!• At the end of each cycle, the amount of DNA has doubled• By the end of 30 cycles, you will have about 1 billion molecules from the original one you started with!! 230=1,073,741,824
  27. 27. The basic protocol—what’s in the tube 5’ 3’Target DNA 3’ 5’ A B Free primers nucleotides Mg2+ Mg 2+ Buffer Taq DNA Mg2+ containing Mg2+ polymerase Mg2+ magnesium Mg2+
  28. 28. 4/28/2012 Free Template from 28
  29. 29. Optimising the PCR Reaction C G Denaturation - - Annealing - Primer extension - -- Ramp - dNTP -DNA Template DNA - PCR - - Tm -
  30. 30. PCR optimization1. For the preparation of reaction mixture, a laminar flow cabinet with UV lamp is recommended2. Fresh gloves should be used for each PCR step3. As well as displacement pipettes with aerosol filters4. The reagents for PCR should be prepared separately and used solely for this purpose5. Aliquots should be stored separately from other DNA samples6. A control reaction (inner control), omitting template DNA, should always be performed, to confirm :a. the absence of contaminationb. or primer multimer formation
  31. 31. Applications of PCR1. the detection of hereditary diseases2. the identification of genetic fingerprints3. the diagnosis of infectious diseases4. the cloning of genes5. paternity testing6. and DNA computing
  32. 32. How It Works • Heating/cooling • Capillary surface area Intake • Single chamber – holds 32 capillaries • Photohybrids measure fluorescence at 530, 640 and 705nm
  33. 33. Virtual PCR Results
  34. 34. Some types of PCR
  35. 35. PCR Cycles
  36. 36. PCR Cycles
  37. 37. PCR Cycles
  38. 38. PCR Cycles
  39. 39. PCR Cycles
  40. 40.  The use of multiple, unique primer sets within a single PCR reaction , to produce amplicons of varying sizes specific to different DNA sequences• By targeting multiple genes at once, additional information may be elicited from a single test run that otherwise : would require several times the reagents and technician time to perform• Annealing temperatures for each of the primer sets , must be optimized to work correctly within a single reaction and amplicon sizes should be separated by enough difference, in final base pair length to form distinct bands via gel electrophoresis
  41. 41. Multiplex PCR• PCR reactions can be devised in which several targets are amplified simultaneously often used in diagnostic applications.
  42. 42. Multiplex pcr
  43. 43. Nested PCRis intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites• Two sets of primers are used in two successive PCR runsthe second set intended to amplify a secondary target within the first run product• This is very successful, but requires more detailed knowledge of the sequences involved
  44. 44. RT-PCR RT-PCR (Reverse Transcription PCR) is the method used to amplify, isolate or identify a known sequence from a cell or tissues RNA library• Essentially normal PCR preceded by transcription by Reverse transcriptase (to convert the RNA to cDNA) this is widely used in :1. expression mapping, determining when and where certain genes are expressed2. detection of RNA viruses
  45. 45. Colony PCRBacterial clones (E.coli) can be screened for the correct ligation products• Selected colonies are picked with a sterile toothpick from an agarose plate, and dabbed into the master mix or sterile water,primers (and the master mix) are addedthe PCR protocol has to be started with an extended time at 95ºC
  46. 46. PCRHot-start PCRTouchdown PCRQuantitative PCRQuantitative real-time PCR 47
  47. 47. Uses of PCR PCR can be used for a broad variety of experiments and analyses:1. Genetic fingerprinting• is a forensic technique used to identify a person by comparing his or her DNA with a given sample• An example is blood from a crime scene being genetically compared to blood from a suspect• The sample may contain only a tiny amount of DNA , (obtained from a source such as blood, semen, saliva, hair, or other organic material)
  48. 48. Uses of PCR2. Detection of hereditary diseases• The detection of hereditary diseases in a given genome is a long and difficult process, : which can be shortened significantly by using PCR• Each gene in question can easily be amplified through PCR by using the appropriate primers : and then sequenced to detect mutations
  49. 49. Uses of PCR3. Viral diseases• can be detected using PCR through amplification of the viral DNA• This analysis is possible right after infection, which can be from several days to several months before actual symptoms occur• Such early diagnoses give physicians a significant lead in treatment• Treatment evaluation, viral load• Genotyping, viruses, bacteria
  50. 50. Uses of PCR4. Mutagenesis• Mutations can be introduced into copied DNA sequences, in two fundamentally different ways in the PCR process• Site-directed mutagenesis allows the experimenter to introduce a mutation at a specific location on the DNA strand• Usually, the desired mutation is incorporated in the primers used for the PCR program• Random mutagenesis, is based on the use of error-prone polymerases in the PCR process the location and nature of the mutations cannot be controlled• One application of random mutagenesis is : to analyze structure-function relationships of a protein• By randomly altering a DNA sequence: one can compare the resulting protein , with the original and determine the function of each part of the protein
  51. 51. Uses of PCR• 5. Genotyping of specific mutations• Through the use of allele-specific PCR, one can easily determine which allele of a mutation or polymorphism an individual has• Here, one of the two primers is common, and would anneal a short distance away from the mutation, while the other anneals right on the variation• The 3 end of the allele-specific primer is modified, to only anneal if it matches one of the alleles
  52. 52. References• ―Polymerase Chain Reaction-Xeroxing DNA‖• ―The Polymerase Chain Reaction‖ ons/pcr.html• ―Polymerase Chain reaction‖• Diagrams from :• Purves, Sadava, Orians, Heller. ―Life.‖ 6th ed. Sinauer Associates, 2001.• ―Mechanism of PCR.‖• “The polymerase Chain Reaction”