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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

  • 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.
  • While many PCR assays are developed for research applications there are further considerations for those that are being developed to become diagnostic assays or to be performed in support of: Biologics License Application (BLA), New Drug Application (NDA), Premarket Approval (PMA), 510(K) or other regulatory filing.
  • Molecular cloning is the process of inserting the gene-of-interest (GOI) into a plasmid vector and this vector is then inserted into a cell that expresses the protein encoded by the GOI. Once protein is expressed in the cell, the protein expression can be used for different studies (such as cell signaling, morphology or other aspects).
  • The Extract-N-Amp™ kits are designed to rapidly extract and amplify genomic DNA. The plant tissue version of these kits has been optimized to amplify without concern over plant inhibitors. This technical document will discuss the versions of this kit that are available and help you find the best kit suited for your needs.
  • Fit-for-use products offer the quality, consistency & documentation necessary for every step of your IVD development and manufacturing process.
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Related Protocols

  • Blue White Screening; DNA Minipreps; Screening by Restriction Digestion; Screening by PCR; Confirm cut plasmid sizes by agarose gel electrophoresis; DNA Maxipreps; DNA Precipitation; RNase Treatment; Clean-up of DNA
  • Whole Genome Amplification: Extraction Protocol - Blood cards provide the convenience of archiving small volumes of blood. However, many times genomic DNA from these samples is limited, which may hinder the researcher’s ability to perform downstream analysis. This protocol provides a simple and convenient method to extract genomic DNA from a blood card.
  • Deoxyribonuclease I is isolated from bovine pancreas and is processed to reduce RNase activity to below detectable levels.
  • The Extract-N-Amp™ Tissue PCR Kit contains all the reagents needed to rapidly extract and amplify genomic DNA from mouse tails and other animal tissues, buccal swabs, hair shafts, and saliva.
  • This protocol provides a simple and convenient method to isolate, amplify and purify genomic DNA from saliva
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