Polymerase Chain Reaction TJey

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PolymeraseChain Reaction

(PCR)

BY,BY,

T.JEYANTHAN T.JEYANTHAN

33RDRD BATCHBATCH



Further«

These include DNA cloning forThese include DNA cloning forsequencing, DNAsequencing, DNA--based phylogeny, orbased phylogeny, orfunctional analysis of genes; thefunctional analysis of genes; the

diagnosis of hereditary diseases; thediagnosis of hereditary diseases; theidentification of genetic fingerprints andidentification of genetic fingerprints andthe detection and diagnosis of infectiousthe detection and diagnosis of infectiousdiseases.diseases.

The method relies on thermal cycling,The method relies on thermal cycling,

consisting of cycles of repeated heatingconsisting of cycles of repeated heatingand cooling of the reaction for DNAand cooling of the reaction for DNAmelting and enzymatic replication of themelting and enzymatic replication of theDNA.DNA.



BASIC PCR

COMPONENTS DNA template that contains the DNADNA template that contains the DNA

region (target) to be amplified.region (target) to be amplified.

Two primers that are complementaryTwo primers that are complementaryto the 3' (three prime) ends of each of to the 3' (three prime) ends of each of the sense and antithe sense and anti--sense strand of thesense strand of theDNA target.DNA target.

TaqTaq polymerase or another DNApolymerase or another DNApolymerase with a temperaturepolymerase with a temperatureoptimum at around 70optimum at around 70 °°C.C.



Cont·d«

DeoxynucleosideDeoxynucleoside triphosphatestriphosphates ((dNTPsdNTPs;;nucleotides containingnucleotides containing triphosphatetriphosphate groups),groups),the building blocks from which the DNAthe building blocks from which the DNApolymerase synthesizes a new DNA strand.polymerase synthesizes a new DNA strand.

Buffer solution, providing a suitable chemicalBuffer solution, providing a suitable chemicalenvironment for optimum activity and stabilityenvironment for optimum activity and stabilityof the DNA polymerase.of the DNA polymerase.

DivalentDivalent cationscations, magnesium or manganese, magnesium or manganeseions; generally Mg2+ is used, but Mn2+ can beions; generally Mg2+ is used, but Mn2+ can beutilized for PCRutilized for PCR--mediated DNA mutagenesis, asmediated DNA mutagenesis, as

higher Mn2+ concentration increases the errorhigher Mn2+ concentration increases the errorrate during DNA synthesis.rate during DNA synthesis.

MonovalentMonovalent cationcation potassium ions.potassium ions.



Equipments

A thermal cycler for PCR

Three-

temperaturethermalcycler

A strip of eightPCR tubes



PCR Procedure

Initialization step (primary construction)Initialization step (primary construction)

DenaturationDenaturation stepstep

Annealing step Annealing step

Extension/Elongation stepExtension/Elongation step

Final elongationFinal elongation

Final holdFinal hold



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««



Procedure mechanism!



Advantages

SensitivitySensitivity

& speed& speed

Less technically difficult.Less technically difficult.



Applications

Comparison of normal cloned geneComparison of normal cloned genewith anwith an uncloneduncloned mutant form of themutant form of the

genegene Detection of low abundance NucleicDetection of low abundance Nucleic

acid sequencesacid sequences

Forensic analysis of DNA samplesForensic analysis of DNA samples

Prenatal diagnosis and carrierPrenatal diagnosis and carrierdetection of cystic fibrosisdetection of cystic fibrosis



Sources

WIKIPEDIAWIKIPEDIA

LIPPINCOTT·S BIO CHEMISTRY LIPPINCOTT·S BIO CHEMISTRY



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Polymerase Chain Reaction TJey

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