Polymerase Chain Reaction (PCR)
- It is an exponential process
- Specifity, Efficacy, Fidelity makes PCR more potent
- Developed in 1983 by kary mullis for which he got Nobel prize in chemistry in 1993
BASIC REQUIREMENT
•
DNA sequence of target region
must be known
•
Primers- typically 20-30 bases
in size. These can be ready produced by
commercial companies. Can also be prepared using a DNA synthesizer
•
Thermo-stable DNA polymerase-
eg Taq polymerase which is not inactivated by heating to 95
•
Deoxy nucleotides (DATP, DGTP,
DTTP, DCTP)
•
Magnesium as a cofactor for Taq
polymerase.
• DNA thermal cycler- machine which can be programmed to carry out heating and cooling of samples of over a number of cycles.
PROCEDURE
•
DENATURATION OF ds DNA
TEMPLATE:- during the heating step (denaturation), the reaction mixture is
heated to 94 for 1 min, which causes separation of DNA double stranded now each
strand act as template for synthesis of complementary strand.
•
ANNEALING OF PRIMERS:- this
step consist of reaction mixture after denaturation step at 54 which causes
hybridization(annealing) of primers to separated strand of DNA(template). The
length and GC content (guanine-cytosine content) of the primer should be sufficient
for stable binding with template. Guanine pairs with cytosine with three
hydrogen bonding adenine binds with thymine with two hydrogen bonds. Thus,
higher GC content is less, length may be increased to have stronger binding
(more number of H bionding between primer and template).
•
EXTENSION OF ds DNA MOLECULES:-
the reaction mixtures heated to 72 which is ideal working temperature for the
Taq polymerase. The polymerase adds nucleotide (dNTP’S) complementary to
template on 3’ -OH pf primers thereby extending the new strand.
• FINAL FOLD:- first three steps are repeated 35-40 times to produce millions of exact copies of the target DNA. Ones several cycles are completed, during the fold step, 4-15C temperature is maintained for short-term storage of the amplified DNA sample.
REACTION CONDITION AND EXPERIMENTAL
PROTOCOL
•
Denaturation condition are best
at 94-95C for 30-60 seconds. Lower temperature may result in incomplete
denaturation of target template denaturation of target template and PCR products.
Higher temperatures and a longer amount of time can lead to enzyme activity
loss.
•
Amount of primer should be
optimum to prevent the formation of primer dimer which is by-products of PCR
reaction
• Contamination should be avoided to prevent foreign genetic material to amplify.
Types of PCR
•
Real-time PCR
•
Quantitative real time PCR
(Q-RT PCR)
•
Reverse Transcriptase PCR
(RT-PCR)
•
Multiplex PCR
•
Nested PCR
APPLICATION
•
Molecular identification
•
DNA Sequencing
•
Genetic engineering
•
DNA fingerprinting
•
Genotyping
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