Polymerase Chain Reaction (PCR) | Molecular Biology

In this article we will discuss about the principle, requirements and procedure for Polymerase Chain Reaction (PCR).

PCR is a simple method for in vitro enzymatic amplification of specific nucleic acids using multiple cycles of template denaturation, primer annealing and primer elongation. From as little as a single piece of target DNA, enough copies can be made for sequencing, cloning or gel electrophoresis. PCR was in­vented by Kary Mullis in 1980 and worked out in 1983.

Principle:

This is a highly sensitive technique used for the detection of minute quantities of nucleic acid in the sample. Two oligonucleotide prim­ers (10 nucleotides) specific to the DNA of interest, and hybridized to opposite strands flanking the target DNA to be amplified, are used in PCR.

Taq polymerase which is a heat stable DNA polymerase, catalyses the elon­gation of the primer. An exponential accu­mulation of a specific DNA fragment occurs due to the respective series of PCR cycles e.g. a 20 cycles of PCR will yield about a million copies of target DNA. The doubling of the number of DNA strands corresponding to the target sequences allows to estimate the am­plification associated with each cycle, using the formula.

amplification = 2ⁿ where ‘n’ is the number of cycles.

Requirements:

1. Taq DNA polymerase:

supplied 30 units, 3 units/µl per reaction store at – 20°C.

2. Deoxynucleotide triphosphates:

This mixture has a final concentration of 2.5 mM of each dNTP and a 10 mM con­centration of total mix store at – 20°C.

3. Assay buffer:

100 µl 10x taq polymer­ase assay buffer with MgCl₂.

Composition:

100 mM tris HCl (pH 9.0), 500 M KC1, 15 mM MgCl₂ and 0.1% gelatin store at -20°C.

4. DNA template:

Genomic DNA purified from Serratia marcescens of approxi­mate concentration 10 mg/µl to be esti­mated. Use 10 µl per reaction.

5. Primer, store at -20°C.

6. Nuclease free water.

7. Mineral oil – 0.5 ml, store at room tem­perature.

8. Gel loading dye – 100 pi, store at 4°C.

9. Agarose:

5 g, store at room temperature.

10. PCR tubes – 12.

11. Control – DNA marker – 5 pg.

12. 50x TAE – 40 ml.

13. Laminar clean air flow.

14. Micropipettes.

15. 0.2 ml microcentrifuge tubes.

Procedure:

Procedure DNA Amplification:

Add this mixture to PCR tubes. Mix gently.

2. Carry out amplification for 30 cycles.

3. When reaction is over, take out reaction mixture and run 10 µl of aqueous layer in 1% agarose gel for 1-2 hours at 100 volt. Run sample along with the marker and locate the amplified product by com­paring with the 0.8 kb fragment of the marker.