Medical Equipment & Automation
specific nucleotide sequencecomposition of that molecule.
Annealing step usually at40
C for 20–40 secondsallowing annealing of theprimers to the single-strandedDNA template. Typically theannealing temperature isabout 3-5
C below the Tm of the primers used. Stable DNA-DNA hydrogen bonds areonly formed when the primersequence very closely matchesthe template sequence. Thepolymerase binds to theprimer-template hybrid andbegins DNA synthesis, and
Extension step at 68
Cwhere thermostable DNApolymerase catalyzes thesynthesis of a new DNAstrand by elongation of the primed strand. Thereaction requires two shortoligonucleotides (primers)flanking the target region to beamplified, which are presentin large molar excess andhybridize to complementarysegments of DNA. During thereaction, deoxynucleotidetriphosphates (dNTP), i.e.,dATP (deoxyadenosinetriphosphate), dCTP(deoxycytidine triphosphate),dGTP (deoxyguanosinetriphosphate) and dTTP(deoxythymidinetriphosphate), are boundto the free 3’-hydroxylend of the new strand.Only deoxynucleotidemonophosphate isincorporated in the DNAchain, cleaving off apyrophasphate group. AsPCR progresses, the DNAgenerated is itself used asa template for replication,setting in motion a chainreaction in which the DNAtemplate is exponentiallyamplified (Fig. 1). The PCR usually consistsof a series of 20-40 repeatedtemperature changes calledcycles; each cycle typicallyconsists of 2-3 discretetemperature steps. Mostcommonly PCR is carriedout with cycles that have threetemperature steps. The cyclingis often preceded by a singletemperature step (called hold)at a high temperature (>90
C),and followed by one hold at theend for final product extension orbrief storage. The temperaturesused and the length of time theyare applied in each cycle dependon a variety of parameters. These
Polymerase chain reaction (PCR) is a technique widely used in molecular biology,biotechnology, microbiology, genetics, diagnostics, clinical laboratories, forensicscience, environmental science, hereditary studies, paternity testing, and manyother applications. It is a primer-mediated enzymatic amplification process ofspecifically cloned or genomic DNA sequences, which was invented by Dr. KaryBanks Mullis who received a Nobel Prize in chemistry in 1993, for his invention ofthe polymerase chain reaction (PCR). This process, which Kary conceptualizedin 1983, is hailed as one of the monumental scientific techniques of the twentiethcentury. It is a method of amplifying DNA; PCR multiplies a single, microscopicstrand of the genetic material billions of times within hours. This technology hasbeen developed in areas as diverse as criminal forensic investigations, foodscience, ecological field studies, and diagnostic medicine.
Fig. 1: Showing three fundamental steps of PCR cycle