PCR AMPLIFICATION
Objective:- To gain hands-on experience of principles and practice of polymerase chain reaction (PCR).
Introduction:- The polymerase chain reaction (PCR) is a DNA amplification technique that has revolutionized almost all aspects of biological research. PCR was invented in 1984 by Dr. Kary Mullis at the Cetus Corporation in California. The enormous utility of the PCR method is based on its ease of use and its ability to allow the amplification of small DNA fragments. For this ground breaking technology, Mullis was awarded the Nobel Prize in Chemistry in 1993.
Principal:-
> Before performing PCR, template DNA is extracted from various biological sources. Because
PCR is very sensitive, only a few copies of the gene are required. Nevertheless, freshly isolated DNA
will provide better amplification results than older DNA specimens that may have become degraded. In
order to amplify the specific DNA or target sequence, two primers (short, synthetic DNA molecules) are
designed to correspond to the ends of the target sequence. The primers hybridize to the DNA template,
which marks this sequence to be copied by DNA polymerase. Starting from the primer, DNA
polymerase builds a new strand of DNA in the 5' ––> 3' direction, using the DNA template as a guide.
> To perform PCR, the template DNA and a molar excess of primers are mixed with the four
“free" deoxynucleotides (dATP, dCTP, dGTP, and dTTP), and a thermostable DNA polymerase. The
most commonly used DNA polymerase is Taq DNA polymerase. This enzyme, originally purified from a bacterium that inhabits hot springs, is stable at very high temperatures. These components (template
DNA, primers, the four deoxynucleotides, and Taq DNA polymerase) are mixed with a buffer that
contains Mg+2, an essential cofactor for Taq polymerase. The PCR reaction mixture is subjected to
sequential heating/cooling cycles at three different temperatures in a thermal cycler.
> In the first step, known as “denaturation", the mixture is heated to near boiling (94°C -96°C) to
“un-zip" (or melt) the target DNA. The high temperature disrupts the hydrogen bonds between the two
complementary DNA strands and causes their separation.
> In the second step, known as “annealing", the reaction mixture is cooled to 45°C - 65°C, which
allows the primers to base pair with the target DNA sequence.
> In the third step, known as “extension", the temperature is raised to 72°C. This is the optimal
temperature at which Taq polymerase can add nucleotides to the hybridized primers to synthesize the
new complementary strands.
> These three steps - denaturation, annealing, and extension - constitute one PCR “cycle". Each
PCR cycle doubles the amount of the target DNA in less than five minutes. In order to produce enough
DNA for analysis, twenty to forty cycles may be required. To simplify this process, a specialized
machine, called a “thermal cycler" or a “PCR machine", was created to rapidly heat and cool the
samples.
Materials and Equipment:-
• PCR kit (MgCl2, PCR buffer, DNA Taq polymerase)
• Thermal Cycler machine
• Pipette
• PCR tubes and tips
• ddH2O
• dNTPmix
• Primer stocks
• DNA sample (from previous practical)
Procedure:-
1. LABEL one 1.5 ml centrifuge tube with "master mix" and your group name or initials.
2. LABEL adequate number of 0.2 PCR tube for different DNA samples in addition to one for negative
control.
3. FILL IN the below table with adequate amount of reagents and multiply based on the number of
samples.
PCR protocol:-
.bmp)
4. MIX the reagents by gently flicking the tube except DNA. This is the PCR master mixture. The
solution should be pale green in color. NOTE: If the solution is not pale green, the PCR sample has
not been correctly assembled.
5. ADD 1 μl of individual DNA samples to 0.2 ml labeled PCR tubes.
6. TRANSFER 9 μl of the PCR master mixture to the labeled 0.2 ml "PCR" tube with already added
DNA.
7. PLACE the "PCR" tube on ice.
8. MIX the sample gently. Make sure the PCR mixture is completely dissolved.
9. Quickly CENTRIFUGE to collect the sample at the bottom of the tube.
10.Recommend appropriate cycling parameters for the PCR machine by filling in the below table:
Cycling parameters:-
.bmp)
10.PLACE the PCR tubes in the PCR machine and RUN.
11.AFTER completing PCR, PLACE the tubes in -20°C for future use.