Polymerase Chain Reaction (PCR)

PCR
Polymerase Chain Reaction is a biochemical technique for in vitro DNA amplification.

PCR was first described by Kary Mullis in 1983..

Polymerase chain reaction

. a process by which DNA is artificially replicated b repeated cycles of an i vitro li t d by t d l f in it DNA polymerase reactions.

The cycling reactions.

There are three major steps in a PCR, which are repeated for 30 to 40 cycles cycles.
1. 1 Denaturation (94 C) . C) 2. Annealing (54 C) C). ( ) 3. extension (72 C) .

Target DNA Sequence 5 3 3 5 3 Annealing 3 5 Denaturation

5 3

Extension

5 3

3 5

The thermal cycler (PCR machine).

The gel electrophoresis appartus

DNA electrophorogram

PCR applications
Cloningofgenes. Hereditarydiseases. y Infectiousdiseases. Geneticfingerprints. Genetic fingerprints Forensicmedicine. Paternitytesting.

PCR components.
DNA template.
One O or more primers. i DNA polymerase (e.g. Taq polymerase).

Deoxynucleotide triphosphates. Buffer solution solution. Divalent cations (Mg+2).

Genetic fingerprinting
UsedtoidentifyapersonbycomparinghisorherDNA withtheDNAinagivensample. g p Comparisonisbasedontheoverallelectrophoretic Comparison is based on the overall electrophoretic patternofDNAfragmentsgeneratedbyrestriction endonucleases digestion of the PCR product digestionofthePCRproduct 'fingerprints'areuniqueexceptforidenticaltwins. fingerprints are unique except for identical twins

Paternity testing
Electrophoresis of PCR-amplified DNA fragments. Genetic relationships can b d G i l i hi be determined b i d by comparing DNA fingerprints. This technique can also be used to determine evolutionary relationships between organisms.

Diagnosis of diseases
Targetgenescanbeamplifiedthensequencedto detectmutations(hereditary). PCRcanbeusedfordetectionofviraldiseases. PCR can be used for detection of viral diseases

AlmostanyinfectiousagentwithaknownDNA Almost any infectious agent with a known DNA sequencecanbeanalyzedbyPCR.

Cloning genes

Cloningagene,describestheprocessofisolatinga Cloning a gene describes the process of isolating a genefromoneorganismandtheninsertingitinto anotherorganism. another organism PCRisusedtoamplifyDNAfragments(genes).

Archeological analysis of fossil.

PCR,toanalyzefossilDNA. y PCRtechniquesforanalysisofafortythousandyear PCR techniques for analysis of a forty thousand year oldDNA(mammoth). TheanalysisofEgyptianmummies.

Genotyping of specific mutations

Determinewhichallele ofamutationor Determine which allele of a mutation or polymorphismexists.

Singlenucleotidepolymorphism.

Comparison of gene expression

QuantitativeversionsofPCRareusedtomonitor Q tit ti i f PCR dt it geneexpression. mRNAisisolatedandreversetranscribedintocDNA. ReversetranscriptasePCR(RTPCR). RealtimePCR(RTPCR)

Reverse Transcription PCR (RT-PCR)

ReverseTranscriptionPCRisthemethodusedto Reverse Transcription PCR is the method used to amplify,isolateoridentifyaknownsequencefrom acellortissuesRNAlibrary. a cell or tissues RNA library EssentiallynormalPCRprecededbytranscriptionby Essentially normal PCR preceded by transcription by reversetranscriptase. Iswidelyusedinexpressionmapping,determining whenandwherecertaingenesareexpressed. when and where certain genes are expressed

Quantitative Real-Time PCR

RealtimePCRisthetechniqueofcollectingdata throughoutthePCRprocessasitoccurs,thus combiningamplificationanddetectionintoasingle step. Thismethodusesfluorescentdyesandprobesto s e od uses uo esce dyes a d p obes o measuretheamountofamplifiedproductinreal time.

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