The specificity of any Real-Time PCR assay, whether TaqMan probe or SYBR Green I, is determined by the quality of the assay design. Non-specific amplification can occur for both SYBR Green I or TaqMan probe methods if the assay design [...]
There are two major detection chemistries used for Real-Time PCR: enzymatic and hydrolysis (TaqMan) probe-based chemistries, and DNA-binding SYBR Green I dye-based chemistry. Additional detection chemistries include Molecular Beacons, Scorpions probes and LUX primers.
There are three phases of PCR amplification: exponential, linear, and plateau. The exponential phase is the first phase of PCR amplification. Reaction components are in excess, there is an exact doubling of product each cycle, and the reaction is specific [...]
Some basic Real-Time PCR terms and their definitions are: Amplification plot – Plot of fluorescent signal versus cycle number. Baseline – The initial cycles of PCR where there is little to no change in fluorescence. Threshold – The arbitrary level [...]
Real-Time PCR has been used in quantification of gene expression, viral quantification, validation of array data, pathogen detection, and allelic discrimination.
One advantage of Real-Time PCR over traditional PCR is that it is a closed-tube system requiring no post-PCR processing. Real-Time PCR has higher precision, increased sensitivity (down to one copy), increased dynamic range (greater than 8 logs), and high resolution [...]
Real-Time PCR uses various fluorescent detection chemistries that allow you to monitor the PCR reaction as it progresses. The amount of fluorescent signal generated is directly proportional to the amount of DNA being synthesized during the PCR reaction. Data are [...]
The starting materials for Real-Time PCR can be RNA, genomic DNA, or plasmid DNA. RNA must be reverse-transcribed into complementary DNA (cDNA) before PCR. More recently, kits have become available that can perform RT-qPCR (RT = reverse transcription) directly from [...]