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In this article we will discuss about:- 1. Meaning of PCR 2. Steps of PCR 3. Applications of PCR.
Meaning of PCR:
In genetic engineering, requirement of desirable amount of target DNA is indispensable to study genetic structure, function and gene manipulation. It is novel cell free techniques to obtain innumerable number of copies. This direct enzymatic amplification process is called the polymerase chain reaction, or PCR. The technique was independently designed and developed by Karymullis in 1980.
In this approach, it is essential to have prior knowledge of a small amount of nucleotide sequence at each end of the regions to be amplified. Based on the knowledge of nucleotide sequence, oligonucleotides complimentary to these are synthesised. These oligonucleotides typically 20 or 50 nucleotides long are known as primers used for enzymatic amplification of target DNA sequence.
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Basic PCR:
A reaction mixture for basic PCR contains the template DNA for amplification, the primers in large concentration of molar excess, dNTPS and a heat-stable DNA polymerase. For this purpose, heat stable Taq polymerase, isolated from Thermus aquaticus, is commonly employed.
The components of the reactions are mixed and then placed in a thermal cycler, which is an automated instrument performs reactions through a series of different temperature for varying nature of time. This series of temperature and time adjustments is referred to as one cycle of amplification.
Each PCR cycle theoretically can double the amount of target DNA sequence. This is otherwise called as amplification. Each cycle of PCR amplification consists of a number of steps. These steps are optimized for each template and primer pair combination. (Fig. 13.12)
Steps of PCR:
Essentially, PCR includes following three steps:
Step 1: Denaturation:
The initial step in a cycle denatures the target DNA by heating reaction mixture to 94°C. At this thermal condition DNA strands gets separated. The reaction temperature is maintained upto 5 minutes.
Step 2: Annealing or Renaturation:
In the second step of a cycle the temperature reduced to approximately 40-60°C. At this temperature, the oligonucleotide primers can form stable association (anneal) with separated target DNA strand and serve as primers for DNA synthesis by a Taq polymerase. This step lasts approximately 30-60 seconds.
Step 3: Synthesis of new DNA:
In the third and final step, synthesis of new DNA begins when the reaction temperature is raised to the optimum for the thermostable DNA polymerase. The temperature is raised to 74°C, extension of primers by the Taq polymerase last approximately 1-2 minutes.
This step completes one cycle and the next cycle begins with a return to 95°C for denaturation. After 20-40 cycles, the amplified nucleic acid may then be analyzed by gel electrophoresis.
Applications of PCR:
PCR is basically employed as diagnostic tool in the identification of specific genetic traits and for the detection of pathogens. In plant biotechnology, for example, preservation of particular plant pathogens can be identified using primers specific for the genomes of the organisms. Detection of gene of interest linked to specific trait can be analysed by PCR.
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Presence of transgene in transformed plants can be screened effectively by PCR method. In addition, diversity of plants, evolution of any origin of particular population or species is being assessed. PCR has also plays an instant role in gene manipulation and expression studies. Development of PCR-based molecular marker has immense potential role in plant breeding and genetic engineering.
