Polymerase chain reaction is a biochemical technique used in Experimental Biotechnology to amplify a specific fragment of target DNA. PCR is a novel molecular procedure based on thermal cycling which consist of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of defined DNA sequences.
PCR was discovered by Kary B. Mulis in 1983 of Cetus Corporation, a Biotech company in California, USA. He won the Nobel Prize for Chemistry in 1993 for ‘contributions to the developments of methods within DNA-based chemistry’. ‘Taq polymerase’ an enzyme used in PCR was described as ‘molecule of the year’ 1989. PCR is now an indispensable technique used in medical and biological research labs for a variety of applications.
PCR was discovered by Kary B. Mulis in 1983 of Cetus Corporation, a Biotech company in California, USA. He won the Nobel Prize for Chemistry in 1993 for ‘contributions to the developments of methods within DNA-based chemistry’. ‘Taq polymerase’ an enzyme used in PCR was described as ‘molecule of the year’ 1989. PCR is now an indispensable technique used in medical and biological research labs for a variety of applications.
A copying machine for DNA molecules
PCR multiplies a single, microscopic strand of the DNA molecule into billions of times within hours. PCR has a major impact on recombinant DNA technology. PCR has multiple applications in medicine, genetics, biotechnology, and forensics.
PCR-A DNA multiplication protocol
PCR is a powerful technique, in which from a single copy of a DNA molecule, millions of copies can be obtained with high accuracy, specificity and in a very short time. DNA amplification process in PCR is cyclical and the concentration of DNA doubles at each cycle. The total amount of DNA concentration increases exponentially during the cyclical process of PCR machine.
PCR-A DNA multiplication protocol
PCR is a powerful technique, in which from a single copy of a DNA molecule, millions of copies can be obtained with high accuracy, specificity and in a very short time. DNA amplification process in PCR is cyclical and the concentration of DNA doubles at each cycle. The total amount of DNA concentration increases exponentially during the cyclical process of PCR machine.
The ‘master mix’ components for PCR machine
· A thermostable DNA polymerase: tag polymerase
· A template DNA
· A complete set of deoxynucleotide triphosphates e.g. dATP, dCTP, dGTP and dTTP
· Tris buffer of pH 8.8
· A pair of oligonucleotide primers
· Mg 2+ and detergents
· 2-mercaptoethanol to stabilize proteins during thermal cycle.
· A template DNA
· A complete set of deoxynucleotide triphosphates e.g. dATP, dCTP, dGTP and dTTP
· Tris buffer of pH 8.8
· A pair of oligonucleotide primers
· Mg 2+ and detergents
· 2-mercaptoethanol to stabilize proteins during thermal cycle.
Requirements for PCR
·
DNA template – DNA segment to be amplified.
· Two primers- a short segment of DNA (forward and
reverse primers) about 20–25 bases long.
· Taq polymerase – an enzyme to synthesize DNA copies.
· Deoxynucleotide triphosphates – the building blocks
for new DNA strand.
· Buffer solution – a suitable chemical environment.
· Divalent cations – Mg 2+ ions
· Monovalent ions
– Potassium ions
· PCR machine – a thermal cycler
Thermostable DNA polymerase
The thermophilic DNA polymerases catalyze template-directed synthesis of DNA from nucleotide triphosphates.
Several thermostable polymerase enzymes are used in PCR
•
Pfu DNA polymerase obtained from - Pyrococcus
furiosus
•
Vent polymerase obtained from- Thermococcus
litoralis
•
Taq polymerase
obtained from - Thermus aquaticus
Oligonucleotide primers
They are synthesized chemically to be complementary to sequences which flank the region of DNA to be amplified. They are usually about 20-25 nucleotides in length. The primers are designed to anneal specifically to the opposite strands of the template molecule. It is the specificity of the primer annealing reaction which ensures that the PCR amplifies the appropriate region of the template DNA.
Critical steps in PCR
1. Sample Preparation
2. Target selection
3.
Primer selection
3 – Temperature cycle in PCR
•
The
temperature - 90-980C separates two strands of target DNA.
•
The
temperature– 40-600C anneals two complementary primers to the ends
of separated single strands of target DNA.
•
The
temperature 720 C allows taq
polymerase to use ss target DNA and primers to synthesize new strands.
PCR protocol
1. Denaturation of ds DNA template –melting of target DNA-it is the thermal denaturation of the dsDNA molecules at 950C for 1 min.
2. Annealing of two oligonucleotide primers at 680C for 60 sec. The annealing temperature is dependent on the length and G+C content of the primer sequences.
3. Extension of dsDNA molecules – temperature is raised to 750C for about 30 sec.
The step cycle programme makes the instrument to heat and cool to the set temperatures due to solid state Peltier-effect device, which actively modulates the desired temperature. There may be as many as 30-35 cycles.
2. Annealing of two oligonucleotide primers at 680C for 60 sec. The annealing temperature is dependent on the length and G+C content of the primer sequences.
3. Extension of dsDNA molecules – temperature is raised to 750C for about 30 sec.
The step cycle programme makes the instrument to heat and cool to the set temperatures due to solid state Peltier-effect device, which actively modulates the desired temperature. There may be as many as 30-35 cycles.
Variants of PCR
· In standard PCR, the sequences of both ends of target
DNA have to be known. Two primers define the ends of target DNA and only that
part is amplified.
· In single sided PCR, the DNA is rearranged before
amplification so that only one primer is needed. This is also called Anchored
PCR.
· In inverse PCR, the DNA at primer sites rather than
between two primers is amplified because primer sites which are bracketing may
have important sequence like promoter for triggering target gene into action.
Characterization of PCR product
•
Contamination
of the reagents by foreign DNA or annealing of primers to alternative sites in
the template DNA may produce unwanted DNA molecules.
•
Multiple
bands in the electropherogram suggest primers annealing to multiple sites.
•
Smear
of DNA suggests presence of excess template DNA.
Problems and limitations
• Contamination of reaction mixture by bacteria, viruses, and our own DNA presents a real problem.
• PCR cannot substitute for cell- based gene cloning, when large amounts of a gene are desired.
• Taq polymerase used in PCR often lack 3' to 5' exonuclease activity. This enzyme lacks the ability to correct mis-incorporated nucleotides.
• PCRs of longer products are less efficient due to enzyme activity loss. PCR can be applied only to short DNA fragments.
• PCR cannot substitute for cell- based gene cloning, when large amounts of a gene are desired.
• Taq polymerase used in PCR often lack 3' to 5' exonuclease activity. This enzyme lacks the ability to correct mis-incorporated nucleotides.
• PCRs of longer products are less efficient due to enzyme activity loss. PCR can be applied only to short DNA fragments.
Applications of PCR
- Detection of pathogens in food, water and tissue specimens.
- Detection of tuberculosis, AIDS and other microbial diseases.
- Diagnosis of genetic diseases-e.g. sickle cell anemia, β-thalasemia, hemophilia
- Identification of criminals, disputed parentage
- Monitor gene expression in genetic engineering or gene therapy experiments.
- To study genetic profile of animals and to trace evolutionary and cultural lineage of human beings.
- To study DNA polymorphism.
- To determine orientation and location of restriction fragments relative to one another.
- To conduct microbial surveillance of the environment.
References
•
Saiki, R., Scharf, S., Faloona, F., Mullis, K.,
Horn, G., and Erlich, H. (1985). Enzymatic amplification of beta-globin genomic
sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 230:
1350-54
•
Mullis, K. and Faloona, F. (1987). Specific
synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods
Enzymol 155: 335-350.
•
Mullis, K. (1990). The unusual origin of the
polymerase chain reaction. Scientific American April 56-65
•
Rabinow, P. (1996). Making PCR: A story of
biotechnology. University of Chicago Press.
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