DNA amplification by PCR

8/3/2019 DNA amplification by PCR

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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 ºc

• 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 ºc

• It allows the primers to anneal with the

templates ( two single strands )

STEP 3: Extension

• Temperature is again raised to 72 ºc

• So, Taq Polymerase adds new nucleotides

and synthesize complimentary strands



Primers :

• Two in numbers

• Complimentary to the targeted DNAsequence at 3’end

• 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 µl )



Taq Polymerase :

• Thermo stable polymerase

• Obtained from “Thermus Aquatiqus”

• Optimum temperature – 72 ºc• Can be stable up to 94 ºc

Some other Thermo stable polymerases

• Thermus thermophilus • Thermotoga martima

• Thermococcus litoralis

• Pyrococcus furiosus

After Step-3 temperature is increased up

to 94º 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

Not sensitive than PCR

Separation of individual DNA clone by comparing

with genomic DNA library can be done

Robustness is not there – amplification can’t done

from material in which DNA

is badly degraded or embedded

Sequence of targeted DNA need not to be known

Proof-reading is possible

Cloning by pcr

Easy and speedy – may take 3-5 min

Very economic,

-Unsophisticated instrument is used

Very sensitive

Minute amount of DNA can be cloned

-Even from single cell

Not done

Robustness-

Amplification 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 agarosegels for detection of PCR amplification

at the final phase of end-point of the

PCR reaction,

• it allow for the detection of PCRamplification during the early phases of

the reaction.

RT-PCR:

Reverse-Transcriptase PCR

For amplification of RNA

RNADNAamplification



Acknowledgement

N.KANAKA DURGA DEVIAsst. Professor

KVSR Siddhartha college of pharmaceutical

sciences,Vijayawada-520010

DNA amplification by PCR

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