Polymerase Chain Reaction (PCR



generating thousands to millions of copies of a particular DNA sequence.Introduction The polymerase chain reaction (PCR) is a scientific technique to amplify a single or a few copies of a piece of DNA across several orders of magnitude. Developed in 1983 by Kary Mullis .

consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. or DNAfunctional analysis of genes.Further« These include DNA cloning for sequencing.based phylogeny. the identification of genetic fingerprints and the detection and diagnosis of infectious diseases. . The method relies on thermal cycling. DNA . the diagnosis of hereditary diseases.

sense strand of the antiDNA target. .BASIC PCR COMPONENTS DNA template that contains the DNA region (target) to be amplified. Taq polymerase or another DNA polymerase with a temperature optimum at around 70 ° C. Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti .

Monovalent cation potassium ions. Buffer solution. but Mn2+ can be utilized for PCR . nucleotides containing triphosphate groups). dNTPs. Divalent cations. . the building blocks from which the DNA polymerase synthesizes a new DNA strand. providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.Cont·d« Deoxynucleoside triphosphates ( dNTPs .mediated DNA mutagenesis. magnesium or manganese cations . generally Mg2+ is used. ions. as PCRhigher Mn2+ concentration increases the error rate during DNA synthesis.

Equipments A thermal cycler for PCR Threetemperature thermal cycler A strip of eight PCR tubes .

PCR Procedure Initialization step (primary construction) Denaturation step Annealing step Extension/Elongation step Final elongation Final hold .

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Procedure mechanism! .

Advantages Sensitivity & speed Less technically difficult. .

Applications Comparison of normal cloned gene with an uncloned mutant form of the gene Detection of low abundance Nucleic acid sequences Forensic analysis of DNA samples Prenatal diagnosis and carrier detection of cystic fibrosis .


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