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.

1. PCR-STEPS, APPLICATIONS,
TYPES OF PCR
Sijo.A
B.Sc.Botany and Biotechnology
Mar Ivanios College,Tvm,Kerala
India

2. 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.

3. 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

4.  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 Taq
polymerase 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.

5. 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.

6. 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

7. 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

9. 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.

10. 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.

12. 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).

13. 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.

15. REFERENCE
1. Dr.U.Satyanarayana; Biotechnology
2. B.D.Singh; Biotechnology