The polymerase chain reaction (PCR) is the cardinal laboratory technology of molecular biology. Arguably one of the most powerful laboratory techniques ever discovered, PCR combines the unique attributes of being very sensitive and specific with a great degree of flexibility. With the PCR it is possible to specifically address a particular DNA Introduction. Polymerase chain reaction (PCR) was invented by Mullis in 1983 and patented in 1985. Its principle is based on the use of DNA polymerase which is an in vitro replication of specific DNA sequences. This method can generate tens of billions of copies of a particular DNA fragment (the sequence of interest, DNA of interest, or target The Polymerase Chain Reaction (PCR) is technique for selective amplification of DNAin vitro. In essence, PCR amplification utilizes the natural machinery of polymerase, the ability to synthesize short oligonu- cleotides, and some sequence knowledge to obtain a pure sample of a specific fragment of DNA. Official Website of Dr. Shyama Prasad Mukherjee University, Ranchi The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment of DNA is amplified using primer mediated enzymes. DNA Polymerase synthesises new strands of DNA complementary to the template DNA. The DNA polymerase can add a nucleotide to the pre-existing 3'-OH group only. Therefore, a primer is required. What is real time PCR? It is a technique used to monitor the progress of a PCR reaction in real-time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified. It is based on the detection of the fluorescence produced by a reporter molecule which increases, as the reaction proceeds. Polymerase chain reaction (PCR) allows the exponential copying of part of a DNA molecule using a DNA polymerase enzyme that is tolerant to elevated temperatures. 1. mRNA is copied to cDNA by reverse transcriptase using an oligo dT primer (random oligomers may also be used). In real-time PCR, we usually use a reverse transcriptase that has an the PCR technique is best demonstrated by reference to specific examples of some of its applications as noted bellow: a) Evolutionary Biology: The PCR analysis of spec-Figure 1: The polymerase chain reaction: (A) the reaction mix-ture and (B) the sequence of events. (c) The third and final stage is an extension reaction PCR steps: PCR step 1: Denaturation PCR step 2: annealing PCR step 3: extension: Time-duration for PCR: PCR reagents: Template DNA: PCR Primer: dNTPs: Taq DNA polymerase: PCR buffer: The PCR machine PCR procedure/ protocol: Key to success in PCR PCR results: Application of PCR Limitations of PCR Types of PCR: Conventional PCR: Gradient PCR: PCR (polymerase chain reaction) is an extremely simple yet immensely powerful technique. It allows enormous amplification of any specific sequence of DNA provided that short sequences either side of it are known. Allow faster diagnosis and identification while enhancing sensitivity and maintaining specificity. Applications of PCR 1. RT-PCR refers to PCR that uses product of an Reverse Transcription (RT) reaction as template 2. A variant of polymerase chain reaction (PCR) 3. A technique commonly used in molecular biology to detect RNA expression 4. RT-PCR is often confused with real-time polymerase chain reaction (qPCR) 5. RT-PCR is used to qualitatively detect gene One primer acts as a sequence-specific primer for first strand cDNA synthesis AND as one of the primers for PCR. The second pri