DNA amplification by PCR

Y. Vijay Surya. KVSR SCOPS

Polymerase Chain Reaction

Invented by Kary B Mullis (Given Noble Prize in 1993) It is a fast, inexpensive and cell free method of DNA cloning.

At the end of this method we get the multiple copies of targeted DNA sequence

Method : Three steps

1.Denaturation 2.Annealing 3.Extension Requirements : DNA that contains sequence of interest Primers OR Oligonucleotides ( 2 in No.) DNA Polymerase Mixture of four deoxynucleoidtriphosphate
Additives : PCR buffer, mgcl2, DMSO, formamide, glycerol, etc..

STEP 1: Denaturation Heat the reaction mixture at 94 c So, double stranded DNA molecule converts in to single stranded DNA ssDNA acts as a template for the primers and DNA polymerase STEP 2: Annealing Reaction mixture is cooled at 50-60 c It allows the primers to anneal with the templates ( two single strands ) STEP 3: Extension

Temperature is again raised to 72 c So, Taq Polymerase adds new nucleotides and synthesize complimentary strands

Primers : Two in numbers Complimentary to the targeted DNA sequence at 3end 15-20 bp long 10-100 picomol of primers required for 100-1000 bp of target sequence Targeted sequence : 100-5,000 bp long (Long PCR allows 42 kbp to be amplified 10-20-10-15 or 1- 105 DNA copies per 100 l )

Taq Polymerase : Thermo stable polymerase Obtained from Thermus Aquatiqus Optimum temperature 72 c Can be stable up to 94 c

Some other Thermo stable polymerases Thermus thermophilus Thermotoga martima Thermococcus litoralis Pyrococcus furiosus
After Step-3 temperature is increased up to 94 c again for denaturation for next cycle After completion of first cycle next 20-35 cycle can perform.

Cell Based cloning Very tedious-it may take weeks Costly compared to PCR

Cloning by pcr Easy and speedy may take 3-5 min Very economic, -Unsophisticated instrument is used

Not sensitive than PCR

Very sensitive Minute amount of DNA can be cloned -Even from single cell
Not done

Separation of individual DNA clone by comparing with genomic DNA library can be done Robustness is not there amplification cant done from material in which DNA is badly degraded or embedded Sequence of targeted DNA need not to be known Proof-reading is possible

RobustnessAmplification can done from material in which DNA is badly degraded or embedded Sequence of targeted DNA must be known Proof-reading is not possible (vent polymerase can be used although not fully efficent ) Sort sized limited amount of product obtained at last

Instrument : Thermal cycler

Applications :
In forensic laboratory in DNA testing To study DNA polymorphisum In molecular mapping In prenatal diagnosis In DNA fingerprinting / In DNA typing In detection of pathogen and disease based on DNA For detection of bacterial and viral infection For monitoring cancer therapy In PCR based diagnostic tests like AIDS, Lyme disease, Hepatitis etc.. For detecting mutations In RNA amplification by RT-PCR In DNA labeling In sexing the embryos

Real-Time PCR:

While traditional PCR uses agarose gels for detection of PCR amplification at the final phase of end-point of the PCR reaction, it allow for the detection of PCR amplification during the early phases of the reaction.
RT-PCR: Reverse-Transcriptase PCR For amplification of RNA RNADNAamplification

Acknowledgement
N.KANAKA DURGA DEVI Asst. Professor

KVSR Siddhartha college of pharmaceutical sciences,Vijayawada-520010