PCR Applications
Polymerase chain reaction (PCR) is a widely used and powerful molecular biology technique that enables the rapid and efficient amplification of specific DNA or RNA sequences from a variety of sources. PCR typically involves target DNA, a pair of synthetic oligonucleotide primers that flank the desired sequence, a thermostable DNA polymerase (such as Taq polymerase), and nucleotides. The process occurs in a thermal cycler and is divided into three main stages during each amplification cycle: denaturation, where double-stranded DNA (dsDNA) is separated into single strands; annealing, where the primers bind to the target sequence; and extension, where the DNA polymerase synthesizes new strands of DNA, effectively creating a copy of the target sequence. After just 20 cycles, this process can produce millions of copies of the original DNA sequence, making PCR a fundamental tool in genetic research, diagnostics, and forensic science.
Overview
Reverse Transcriptase PCR (RT-PCR)
RT-PCR, or reverse transcriptase PCR, is a variation of the standard PCR technique that involves the amplification of specific mRNA obtained from very small samples. It eliminates the need for the tedious mRNA purification process required for conventional cloning techniques. With RT-PCR, reverse transcriptase and an RNA sample are used in addition to the standard PCR reagents. The reaction mixture is heated to 37 ˚C, which allows for the production of complementary cDNA copy from the RNA sample by reverse transcriptase. This cDNA then anneals to one of the primers leading to first strand synthesis. Standard PCR follows from here in which dsDNA is ultimately generated. RT-PCR is frequently combined with real time PCR (qPCR), which is widely used for the quantification of transcript levels in cells and tissues.
Hot Start PCR
Hot Start PCR is a technology that inhibits hot start Taq polymerase or the incorporation of modified dNTPs during reaction set up until a heat activation step occurs. Various methods are available to arrest hot start polymerase activity and include, chemical modification, antibody-mediated, and aptamer-mediated methods.
Endpoint PCR for Long and Accurate Target Amplification
Endpoint PCR is often used to detect the presence of targets and relative abundance at the completion of the reaction. The limited length of sequences produced during standard PCR, approximately 5 kb, is in part overcome with the incorporation of additional factors that provide “proofreading” activity. Long and accurate (LA) PCR incorporates the use of a second thermostable polymerase with 3′→5′ exonuclease to repair terminal nucleotide misincorporations, resulting in significantly increased fidelity and the ability to amplify DNA targets up to 40 kb in length.