MilliporeSigma
HomeApplicationsGenomicsPolymerase Chain Reaction Applications

Polymerase Chain Reaction (PCR) Applications

Reverse transcriptase (RT) PCR follows these steps: isolation of RNA or mRNA, primer annealing, first strand synthesis, and PCR amplification

Polymerase chain reaction (PCR) is a powerful core molecular biology technique that is an efficient and rapid in vitro method for enzymatic amplification of specific DNA or RNA sequences from various sources. A standard PCR consists of target DNA, a set of synthetic oligonucleotide primers that flank the target DNA sequence, a thermostable DNA polymerase (usually Taq polymerase), and nucleotides. Using thermal cyclers, there are three stages during each amplification cycle, including denaturing double-stranded DNA (dsDNA) into separate single stranded DNA, annealing primers to the target DNA sequence, and extension, where DNA polymerase extends the DNA from the primers, creating new dsDNA with one old strand and one new strand. The strands synthesized in one cycle serve as a template in the next, resulting in a million-fold increase in the amount of DNA in just 20 cycles.

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.


Related Technical Articles

  • This page shows PCR and RT-PCR amplification troubleshooting.
  • KOD One™ PCR Master Mix overview for ultra-fast PCR with high specificity, fidelity, and yield
  • A PCR master mix is a batch of PCR or RT-PCR reagents that can be divided among many PCR reaction tubes. It usually includes DNA polymerase, dNTPs, MgCl2 and buffer. Make your own master mix or choose a commercial one.
  • Developing a PCR or RT-PCR/RT-qPCR troubleshooting protocol so that data are reliable is essential. Potential sources of RT-PCR or PCR error and problems include operator error, the PCR master mix, and oligo design. This PCR troubleshooting guide outlines and details fixes for your RT-PCR assay.
  • The polymerase chain reaction is one of the most widely used techniques in molecular biology. The PCR process consists of three main steps, Denaturation, Annealing & Extension
  • See All

Related Protocols

  • Learn standard PCR protocol steps and review reagent lists or cycling parameters. This method for routine PCR amplification of DNA uses standard Taq DNA polymerase.
  • Annealing is the process of heating and cooling two single-stranded oligonucleotides with complementary sequences. Heat breaks all hydrogen bonds, and cooling allows new bonds to form between the sequences.
  • Calculate the melting temperature (Tm) of oligonucleotides using theoretical method like the nearest neighbor or the basic method. The DNA Tm is the temperature at which 50% of the oligonucleotide is duplexed with its perfect complement and 50% is free in solution.
  • The entire PCR workflow is vulnerable to factors which introduce variability. Many of the variable components are unavoidable, such as the source of the sample or the requirement for a reverse transcription step. Assay design is also highly variable and can make the difference between PCR success and failure and also contributes to the reproducibility and sensitivity of an assay.
  • The exrract-N-Amp kit protocol provides a rapid DNA extraction method that leads to a PCR-ready sample in 15 minutes. Optimized PCR ReadyMix yields successful and clean amplification. RED loading dye allows for sample tracking.
  • See All