MyBioSource PCR Kit
|The powerful polymerase chain reaction (PCR) molecular biology technique enables scientists to hone in on a segment of DNA, and copy it thousands, millions or even billions of times over. This can be accomplished relatively easily and inexpensively. It is incredible that a single copy or just a few copies of a DNA molecular sequence can can be amplified into thousands or more copies of the same DNA molecule.|
|Since it was first developed in 1983 by Kary Mullis who won the nobel prize in Chemistry in 1993 for his invention, PCR has become an invaluable molecular biology technique. Today PCR tools, reagents, and kits are used worldwide for a variety of applications. This includes identification of pathogens and infectious agents, disease diagnosis, analysis of genetic and hereditary conditions, and cloning DNA fragments for insertion into vectors, to name a few.|
|In conventional PCR, the amount of accumulated PCR product is measured at the end of PCR cycles after the reaction is complete. In this traditional approach, it is not possible to determine the starting concentration of the target DNA since results are only obtained after the reaction is finished. However, the PCR amplified product can be run on an agarose gel and the intensity of the band can be compared to standards of known concentrations to obtain a semi-quantitative result. Conventional PCR amplification of DNA is widely used for cloning, sequencing and genotyping.|
In contrast to the traditional technology, real time PCR in relatively quantitative.
|Real time PCR, also known as the quantitative polymerase chain reaction (qPCR), measures amplification of the target DNA molecule during the reaction as the amplification is occurring. Data is continuously collected during the log/exponential phase of the PCR reaction when products are doubling at every PCR cycle. During this phase, the quantity of the PCR product is directly proportional to the amount of starting template DNA. qPCR is among the most sensitive genomic analysis technique available. Applications are broad, including pathogen detection, genotyping, viral quantitation, and quantitative gene expression analysis.|
|Another variation of traditional PCR is the reverse transcription polymerase chain reaction (RT-PCR). RT-PCR is used to detect gene expression through generating complimentary DNA (cDNA) transcripts from RNA. In RT-PCR, reverse transcriptase is used to reverse transcribe the RNA of interest into its complement DNA. The newly synthesized cDNA is then amplified by PCR. qPCR can be incorporated into the technique and the combined technique is described as quantitative real time PCR, real time RT-PCR or qRT-PCR.|
MyBioSource is a leading PCR solution Kit provider.
|Real time PCR (qPCR) and real time RT-PCR (qRT-PCR) ready to use Kits are popular among scientists because the design, including primer design for PCR amplification, positive controls and other kit components have already been optimized for detection.
|Detailed methodology is explained in the Kit manuals. In brief, for many of the qPCR kits detection is based on the fluorogenic 5'nuclease assay. In this assay, DNA polymerase cleaves the primer-probe at the 5' end and separates the reporter dye from the quencher dye only when the probe binds or hybridizes to the target DNA. The cleaved reporter dye generates a fluorescent signal which is monitored in real time by the PCR detection system and the increase in fluorescence signal is proportional to the amount of PCR product. For RT-PCR, the mRNA is first transcribed into cDNA, and then a thermostable DNA polymerase is used to amplify the specific, target gene fragments by PCR.|
Kits are available for infectious diseases, animal pathogens, tumor detection, genetic diseases as well as for monitoring pathogens associated with organ transplantation.
|Many of the kits are compliant with ISO13485 and CE marked. The kits are compatible with most brands of real time PCR systems including Roche, BioRad, and Applied Biosystems.|