PCR techniques

From OpenWetWare

Revision as of 17:06, 9 October 2007 by Reshma P. Shetty (Talk | contribs)
Jump to: navigation, search

Several techniques have have been derived from the basic polymerase chain reaction (PCR). Below is an overiew of important PCR methods linking to individual pages for detailed information.

  • nested PCR is 2 successive PCRs with the 2nd set of primers nested inside the 1st pair. It is used to reduce unspecific products.
  • multiplex PCR is a PCR with >1 primer pair run in a single reaction. It reduced material consumption but is hard to optimise. It is often used in mouse genotyping for example.
  • colony PCR is a PCR technique to detect DNA in bacterial colonies. Colonies are picked, lysed, and amplified by PCR. It is used to detect successful ligations or recombinations among large numbers of bacterial colonies.
  • RT-PCR (Reverse Transcription PCR) is essentially normal PCR preceded by a reverse transcription converting RNA to cDNA. It is used to indirectly detect/measure RNAs, e.g. messenger RNAs. Confusingly, RT PCR is used as an abbreviation for real-time PCR.
  • Q-PCR (quantitative PCR) is used to determine the quantity of starting nucleic acid template. There are 2 main methods: PCR with dsDNA dyes and PCR with probes. dsDNA dyes and probes allow measurement while the PCR is running, i.e. in real-time, and are therefore often named real-time PCR (see below). Probes are superior to dsDNA dyes but also more expensive. DNA can also be quantified with a simple PCR followed by agarose gel electrophoresis (qPCR end-point assay). If done properly with serial dilutions of starting DNA template to establish a linear relationship between band intensity and starting material, this approach is also quantitative.
  • Real-time PCR is used to determine the quantity of DNA real-time and thus a subset of the Q-PCR methods. It uses fluorescent dyes or fluorophore-DNA probes to measure the amount of amplification in real time. This is used to infer starting amount. Real-time PCR is often confusingly abbreviated as RT PCR which overlaps with reverse transcription PCR.
  • Quantitative RT-PCR or quantitative reverse transcription PCR refers to quantitative PCR of cDNA (which has been reverse transcribed from RNA). Like quantitative PCR, you can quantify the starting RNA template either during the reaction ("Real-time reverse transcription PCR") or at the end of the PCR step.


Personal tools