2 WORKSHOP (1) Presented by: Afsaneh Bazgir Polymerase Chain Reaction (Msc Student of Human Genetics)Golestan University of Medical Sciences(GOUMS)
3 DefinitionPolymerase Chain Reaction (PCR): A technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across generating thousands to millions of copies of a particular DNA sequence.Developed in 1983 by Kary Mullis.In 1993, Mullis was awarded the Nobel Prize in Chemistry along for his work on PCR.
4 Applications DNA cloning for sequencing DNA-based phylogeny, or analysis of genesThe diagnosis of hereditary diseasesThe identification of genetic fingerprints (used in forensic Sciences and paternity testing)Detection and diagnosis of infectious diseases
5 Most PCR methods typically amplify DNA fragments of between 0.1 and 10 kbp, although some techniques allow for amplificationof fragments up to 40 kbp in size.The PCR is commonly carried out in a reaction volume of10–200 μl in small reaction tubes.The thermal cycler (thermocycler) is a laboratory apparatus mostcommonly used to amplify segments of DNA via the PCR.
8 DNA template: Primers: Taq polymerase : Basic requirements for PCR set upDNA template: contains the DNA region (target) to be amplified.Primers: complementary to the 3' ends of each of the strands of the DNA target.Taq polymerase :With a temperature optimum at around 70 °C.Purified from the thermophilic bacterium, Thermus aquaticus, whichnaturally lives in hot (50 to 80 °C (122 to 176 °F)) environments.
9 Deoxynucleoside triphosphates (dNTPs): Cont…Deoxynucleoside triphosphates (dNTPs):The building-blocks from which the DNA polymerase synthesizes a newDNA strand.Buffer solution:Providing a suitable chemical environment for optimum activity and stability ofthe DNA polymerase.Bivalent cations: Magnesium or Manganese ions; generally Mg2+ is used, but Mn2+ canbe utilized for PCR-mediated DNA mutagenesis, as higherMn2+ concentration increases the error rate during DNA synthesis.
10 Procedure 1. Denaturation step 2. Annealing step PCR consists of a series of repeated temperature changes, with eachcycle commonly consisting of 3 temperature steps:1. Denaturation step2. Annealing step3. Extension/elongation stepFinal elongationFinal hold
11 It causes DNA melting of the DNA template by disrupting the hydrogen Denaturation stepThe first regular cycling event, consists of heating the reaction to 94–98 °Cfor 20–30 seconds.It causes DNA melting of the DNA template by disrupting the hydrogenbonds between complementary bases.Yielding single-stranded DNA molecules.ONE
13 Allowing annealing of the primers to the single-stranded DNA template. Annealing stepThe temperature is lowered to 50–65 °C for 20–40 seconds.Allowing annealing of the primers to the single-stranded DNA template.This temperature needs to be low enough to allow for hybridization of theprimer to the strand, but high enough in order for the hybridization to bespecific.TWO
14 the primer might not bind. Cont…If the temperature is too low, the primer could bind imperfectly. If it is too high,the primer might not bind.Stable DNA–DNA hydrogen bonds are only formed when the primer sequencevery closely matches the template sequence.The polymerase binds to the primer-template hybrid and begins DNA formation.
16 Taq polymerase has its optimum activity temperature at 75-80°C and Extension/elongation stepThe temperature at this step depends on the DNA polymerase used. Taq polymerase has its optimum activity temperature at 75-80°C andcommonly a temperature of 72 °C is used with this enzyme.At this step the DNA polymerase synthesizes a new DNA strandcomplementary to the DNA template strand by adding dNTPs thatare complementary to the template in 5' to 3' direction.THREE
17 Cont…The extension time depends both on the DNA polymerase used and on thelength of the DNA fragment to be amplified.At its optimum temperature, the DNA polymerase will polymerize a thousandbases per minute.Under optimum conditions, (if there are no limitations due to limitingsubstrates or reagents), at each extension step, the amount of DNA targetis doubled, leading to exponential amplification of the specific DNA fragment.
19 Final elongationThis single step is occasionally performed at a temperature of 70–74 °C.This is the temperature needed for optimal activity for most polymerases usedin PCR for 5–15 minutes after the last PCR cycle to ensure that any remainingsingle-stranded DNA is fully extended.Final holdThis step at 4–15 °C for an indefinite time may be employed forshort-term storage of the reaction.