PCR-STEPS, APPLICATIONS,

TYPES OF PCRSijo.A

B.Sc.Botany and Biotechnology

Mar Ivanios College,Tvm,Kerala

India

PCR

The term PCR stands for Polymerase Chain Reaction.

It is an invitro amplification technique that allows synthesizing millions of copies of the DNA or gene of interest from a single copy.

It is called “Polymerase” because the only enzyme used in this reaction is DNA polymerase.

The PCR is invented by Kary Mullis in 1985.He received Nobel Prize in Chemistry in 1993.

REQUIREMENTS OR REACTION

COMPONENTS

1. Target DNA

contains sequence to be amplified.

2. Two oligonucleotide primers

Binds to the 3’OH end of both strands.

3. dNTP ( deoxy Nucleotide TriPhosphates)

DNA building units.

They are four kinds: dATP, dTTP, dGTP, DCTP

4. Heat stable DNA polymerase

i) Taq DNA Polymerase - isolated from Thermus

aquaticus.

ii) Pfu Polymerase - Pyrococcus furiosus.

iii) vent Polymerase – Thermococcus litoralis

Taq DNA polymerase lacks proof reading activity(3’5’), so it commits error at a high rate of 2x10-4.

pfu and vent polymerase are more efficient than Taqpolymerase because it have proof reading activity.

so they commits error at a lower rates of 4x10-5.

5. Bivalent cations like Mg++ and Mn++ ions

cofactor of the polymerase enzyme.

6. Buffer solution

they maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme.

STEPS INVOLVED IN PCR

The amplification is achieved by three step process.

1. Denaturation (90-98 °C)

2. Renaturation or primer annealing (40-60 °C)

3. Extension (70-75 °C)

o The steps are automated.

o one cycle lasts for 3minutes to 45 seconds.

DENATURATION

Denaturation is the first step and it involves thermal denaturation of DNA sample by raising the temperature to

Temperature denatures dsDNA into two single strands by breaking H-bonds between them.

RENATURATION OR PRIMER ANNEALING

It is the second step where the temperature is brought down to

The annealing of primer to the 3’-end of target DNA at both strands takes place.

The annealing temperature depends on the length and base sequence of a primer being used for amplification.

The duration of annealing is usually 1 minute.

95 °C

60 °C

EXTENSION

It is the step where the temperature is raised to , which is optimum for the catalytic functioning of Taq DNA Polymerase.

DNA synthesis is initiated from 3’-end by the addition of nucleotides by Taq DNA Polymerase.

72°C

APPLICATIONS

1. To amplify DNA fragments isolated from organism.

2. To propagate DNA for gene manipulation and DNA libraries.

3. Used in sex determination of embryo.

4. PCR products can be used for mapping genes.

5. PCR products can be used as probes.

6. Used to detect genetic diseases.

7. Used to sequence DNA directly.

8. Used in Forensic science and DNA finger printing.

RT-PCR

RT-PCR denotes Reverse Transcriptase PCR.

It is used for the amplification of RNA molecules.

For this purpose RNA molecule (mRNA) is converted into complementary DNA (cDNA) by the enzyme reverse transcriptase.

Then cDNA serve as a template for PCR.

Different primers can be used for the synthesis of first strand of DNA.

It includes Random primer, Oligo dTprimer and Sequence specific primer.

REAL TIME PCR

Real Time PCR is also known as quantitative Polymerase Chain Reaction (qPCR).

It monitors the amplification of targeted DNA molecule during the PCR.

Mainly there are two ways for the synthesis of product in real time.

1. Non specific fluorescent dyes that intercalate with any double stranded DNA.

SYBR Green is an asymmetrical cyanine dye that will bind to dsDNA and form dye complex.

They absorbs blue light (497nm) and emits green light (520nm).

REAL TIME PCR

2. Sequence specific DNA probes consisting of short oligonucleotide called reporter probe.

They give fluorescent signal after the hybridization of PCR product by probe.

An oligonucleotide consists of fluorescent dye and quenching compound inhibits flourescent signal.

The hybridisation between oligonucleotide and PCR product disrupts the base pairing and moving quencher away from the dye and flourescent signal is generated.

REFERENCE

1. Dr.U.Satyanarayana; Biotechnology

2. B.D.Singh; Biotechnology