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Multiplex PCR
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multiplex PCR, a variant of the test in which more than o ne target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described.The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids.) Quantitative polymerase chain reaction From Wikipedia, the free encyclopedia (Redirected from QPCR ) Jump to: navigation , search Quantitative polymerase chain reaction (qPCR) is a modification of the polymerase chain reaction used to rapidly measure the quantity of DNA , complementary DNA or ribonucleic acid present in a sample. Like other forms of polymerase chain reaction, the process is used to amplify DNA samples, via the temperature-mediat ed enzyme DNA polymerase. PCR theoretically amplifies DNA exponentially, doubling the number of molecules present with each amplification cycle. The number of amplification cycles and the amount of PCR end-product should allow one to calculate the initial quantity of genetic material, but numerous factors complicate this calculation. The ethidium bromide staining typically used to assess a successful PCR prevents further amplification, and is only semi-quantitative. The polymerase chain reaction may not be exponential for the first several cycles, and furthermore, plateaus eventually, so care must be taken to measure the final amount of DNA while the reaction is still in the exponential growth phase. To overcome these difficulties, several different quantitative methods have been developed. The most sensitive quantification methods are done by the real-time polymerase chain r eaction , where the amount of DNA is measured after each cycle of PCR by use of fluorescent markers. Other end-point methods measure DNA after PCR is completed. These methods depend on addition of a competitor RNA (for reverse-transcriptase PCR ) or DNA in serial dilutions or co-amplification of an internal control to ensure that the amplification is stopped while in the exponential growth phase. Although real-time quantitative polymerase chain reaction is often marketed as RT-PCR, it should not to be confused with reverse transcription polymerase chain reaction , which is also referred to as RT-PCR, but is used to amplify RNA samples. The two methods may be used in concert to reverse transcribe RNA and then quantitate the resulting cDNA using real-time PCR (often referred to as real-time RT-PCR). Quantitative Real-Time Polymerase Chain Reaction for the Core Facility Using TaqMan and the Perkin-Elmer/ Applied Biosystems Division 7700 Sequence Detector Deborah S. Grove Nucleic Acid Facility, Life Science Consortium, The Pennsylvania State University, Universit y Park, PA 1680 2 Quantitative real-time polymerase chain reaction (PCR) using the Perkin Elmer/Applied Biosystems Division 7700 Sequence Detector provides an accurate method for determination of levels of specific DNA and RNA sequences in tissue samples. It is based on detection of a fluorescent signal produced p roportionally during amplification of a PCR product. Turn-around time for data acquisition and analysis by real-time PCR with the 7700 model is short, and results are more reliable than by traditional PCR methods. This technology can be successfully offered as a service in a core faculty setting. (J Biomol Tech 1999;10:11-16) Key words: quantitative real-time polymerase chain reaction, Perkin Elmer/Applied Biosystems 7700 Sequence Detector, nucleic acid sequence quantification.
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