Polymerase Chain Reaction (PCR)

1. Polymerase chain reaction (PCR)

2. • The Polymerase chain reaction (PCR) is a laboratory (in vitro)
technique for generating large quantities of specified DNA.
• PCR is a cell-free amplification technique for synthesizing multiple
identical copies (billions) of any DNA of interest.
• Developed in 1984 by Kerry Mullis.
• PCR is now considered as a basic tool for the molecular biologist.
Polymerase chain reaction (PCR)

3. Principle of PCR
• The double-stranded DNA of interest is denatured to separate into two
individual strands.
• Each strand is then allowed to hybridize with a primer (renaturation).
• The primer-template duplex is used for DNA synthesis (the enzyme-
DNA polymerase).
• These three steps-- denaturation, renaturation and synthesis are
repeated again and again to generate multiple copies of target DNA.

4. The essential requirementsfor PCR are listed below:
• A target DNA (100-35000 bp in length)
• Two primers (synthetic oligonucleotides of 17-30 nucleotides in length)
that are complementary to regions flanking the target DNA.
• Four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP)
• A DNA polymerase that can withstand at a temperature up to 95°c(i.e.;
thermo stable)
The reaction mixture contains the target DNA, two primers (in excess), a
thermo stable DNA polymerase (isolated from the bacterium Thermus
aquaticus (i.e., Taq DNA polymerase) and four deoxyribonucleotides

5. Steps in PCR
• The actual technique of PCR involves repeated cycles for amplification
of target DNA. Each cycle has three stages.
– Denaturation of template dsDNA
– Renaturation or annealing of primers
– Synthesis of complementary strand

6. • Denaturation: On raising the temperature to about 95°C for about one
minute, the DNA gets denatured and the two strands separate.

7. • Renaturation or annealing: As the temperature of the mixture is slowly
cooled to about 55°C, the primers base pair with the complementary
regions flanking target DNA strands. This process is called
renaturation or annealing. High concentration of primer ensures
annealing between each DNA strand and the primer rather than the
two DNA strands

8. • Synthesis: The initiation of DNA synthesis occurs at 3'-hydroxyl end of
each primer. The primers are extended by joining the bases
complementary to DNA strands. The synthetic process in PCR is quite
comparable to the DNA replication of the leading strand. However the
temperature has to be kept optimal as required by the enzyme DNA
polymerase. For Taq DNA polymerase, the optimum temperature is
around 75°C (for E. Coli DNA polymerase, it is around 37°C). The
reaction can be stopped by raising the temperature (to about 95°C).

10. The three stages of PCR in relation to temperature and time are depicted
in figure below:
Each cycle of PCR takes about 3-5 minutes. In normal practice, the PCR is
carried out in an automated machine, Thermo cycler.

11. Key factors for optimal PCR
• Primers: Primers play a significant role in determining PCR. The
primers (17-30 nucleotides) without secondary structure and without
complementarity among themselves are ideal. The complementary
primers can hybridize to form primer dimer and get amplified in PCR.
This prevents the multiplication of target DNA

12. • DNA polymerase: A DNA polymerase that can withstand at a
temperature up to 95°C (i.e.; thermo stable) is essential. Taq DNA
polymerase is preferred as it can withstand high temperature.
However, Taq polymerase lacks proof reading exonuclease (3'-5')
activity which might contribute to errors in the products of PCR. Some
other thermo stable DNA polymerases with proof reading activity have
been identified e.g., Tma DNA polymerase from Thermotoga
maritama; Pfu DNA polymerase from Pyrococcus furiosus.

13. • Target DNA: In general, the shorter the sequence of target DNA, the
better is the efficiency of PCR. However in recent years, amplification
of DNA fragments up to 10 kb has been reported. The sequence of
target DNA is also important in PCR. Thus, GC rich regions of DNA
strand hinder PCR.
• Promoters and inhibitors: Addition of proteins such as bovine serum
albumin (BSA) enhance PCR by protecting the enzyme DNA
polymerase. Humic acids, frequently found in archaeological samples
of target DNA inhibit PCR.

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