qPCR / Real-Time PCR
Real-time PCR (or qPCR) proceeds in the same way as the standard polymerase chain reaction technique, but the concentration of the target DNA sequence is observed and/or recorded in real time, rather than just at the end of the amplification process. Small amounts of messenger RNA (mRNA), which regulates gene expression in the cell, can be also amplified, once the RNA has been reverse...
...transcribed to complementary DNA (cDNA). Real-time PCR techniques can be used to determine relative gene expression, quantification and/or genotyping of infectious diseases, viruses, GMOs, and pathogens in agriculture, water supplies, and food & beverages.
There are two basic methods of tracking the PCR process in real time using optical detectors. One is to use non-specific fluorescent dyes, which bind with any double-stranded DNA molecule. Another is to use “reporter” probes consisting of an oligonucleotide complementary to the target sequence at a point between the two primers. The probe contains a linked fluorescent dye molecule and a quencher molecule that inhibits fluorescence. During the polymerization process, the probe specifically binds within the target DNA sequence at the same time as the PCR primers. But once the sequencing action of the polymerase reaches the bound probe, the fluorescent molecule is knocked off, causing an increased fluorescence which can be read. As the sequencing continues along the DNA strand, the probe releases the quencher into solution, and causing a drop in the fluorescence reading. Thus, the technician can “see” and measure the results of each cycle of the DNA amplification as it progresses in real time.
Biochemicals and reagents for qPCR include custom oligonucleotides and gene fragments; pPCR primers and assays; Taq polymerase; probes; real-time PCR kits with different probe chemistries; cell lysing kits; high-throughput fragment library prep solution; transfection reagent; RNA extraction solution; DNA removal enzyme kits; and universal microRNA expression profiling panels and assays.
Instrumentation and equipment for qPCR include real-time PCR detection systems; automated qPCR prep systems; PCR plates and stands; PCR tube strips; heat sealing systems and seals; LIMS systems; single- and multi-channel pipettes; microvolume cuvettes; tissue homogenizers; sample coolers; centrifuges; and nucleic acid analysis systems.
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