AIM: To study the protocol of Polymerase Chain Reaction (PCR)
Requirements: PCR components: Basic PCR reaction requires four components:
(1) DNA template.
(2) Primers.
(3) Deoxynucleotide triphosphates (dNTPs).
(4) Thermostable DNA polymerase.
(5) Thermal cycler machine
(6) Buffer solution
Basic PCR components and their functions
Components Function
Template DNA The template carries the DNA segment or (target) component to be amplify
Forward and A primer is a short, single-stranded piece of DNA that anneals (attaches) to
Reverse Primers its complementary sequence on the template. A pair of primers will bind to
either side of the target DNA segment providing initiation sites for DNA
synthesis.
DNA This is the enzyme used to synthesize new strands of DNA. The DNA
polymerase polymerase adds nucleotides onto the end of an annealed primer.
dNTPs These are the four nucleotides used by DNA polymerase to extend an
annealed primer (building blocks).
Magnesium DNA polymerase requires magnesium for activity (co-factor). Magnesium
is usually supplied to a PCR amplification in the form of magnesium
chloride.
PCR buffer PCR buffer is necessary to create optimal conditions for activity of Taq
DNA polymerase.
Thermal cycler To perform the heating and cooling cycles
Theory: Polymerase chain reaction (PCR), a process of DNA replication in cell where particular
piece of DNA can be amplified in billions of copies in a short time. The PCR amplify a precise
fragment of DNA from a complex mixture of starting material termed the template DNA which
controlled by heating and cooling. It is an important process in biotechnology and molecular
biology and has been widely used in research, medicine, agriculture and forensics.
PCR cycle: PCR proceeds in THREE distinct steps Governed by Temperature:
Procedure of PCR
It is a multistep process which consists of following steps:
Initialization: It consists of heating the reaction chamber to a temperature of 94– 96 °C (94 °C)
for 1 to 5 min. It causes separation of DNA double helix – Initial denaturation.
Denaturation: It is the first regular cycling event which consists of heating the reaction chamber
to a temperature of 94–98 °C for 20 – 30 seconds. It causes melting, or denaturation of DNA by
breaking the hydrogen bonds between complimentary bases, yielding two single-stranded DNA
molecules. Each strand acts as a template for synthesis of complimentary strand.
Annealing: Temp is lowered to 50 – 65 °C and kept the reaction mixture at this temp for 20 – 30
sec. This allows hybridization of primers to the primary strand.
Extension/Elongation: Reaction chamber is heated to 72 °C for 1 min. In this step, Taq DNA
polymerase synthesize a new DNA stand complimentary to the DNA template strand by adding
free dNTPs from the reaction mixture.
Final extension: This step is optional. It is performed at a temperature of 70 – 74 °C (72 °C) for
7 -10 min. It ensures that any remaining single-stranded DNA is fully elongated.
Store: Now the final product is stored for analysis. Generally stored at 4 – 15 °C
Calculation of copies in PCR :
The number of double stranded DNA pieces is doubled in each cycle, so that after n cycles you
have 2n (2 to the nth power) copies of DNA.
