PCR Assay Designby Fluoresentric
Genotyping is used to separate individuals based on genetic differences. When designing a genotyping assay for PCR, we look at specific single nucleotide polymorphisms (SNPs) and run melt analyses. These designs must be very specific and sensitive in order to clearly separate individuals based on a single SNP.
Multiplex PCR is used to detect two areas of interest in the same reaction. For a duplex, two sets of primers and probes are added into the PCR reaction for two different targets on the same template. For a triplex, three sets of primers and probes are added, and so on. This has the potential to reduce costs because fewer reagents are needed and fewer reactions need to be run. Multiplex assays can become extremely difficult to design and optimize because each of the primer sets must have similar annealing and melting temperatures and must not have cross-complementarity.
Quantitative PCR is used to determine how much of specific target is in a sample, such as how many yeast bacteria are in a specific volume of liquid. Quantification is based on a standard curve for the reaction and how many cycles it takes to reach a detectable fluorescence in a real-time PCR machine. The fewer cycles it takes to amplify, the greater the amount of starting copies of material.
Conventional PCR, or Traditional PCR, is PCR that does not use real-time instruments, but relies on gels to determine if you have amplified product. Real-time PCR is superior to conventional PCR because it is quantitative and it measures amplification as it happens. Conventional PCR has the advantage of separating products out on a gel based on size.
TaqMan probes are real-time PCR hydrolysis probes that use the Taq polymerase to cleave a the probe bound to the desired template. When the probe is cleaved, the fluorophore separates from a quencher and provides the signal. TaqMan probes can be used for gene expression, template detection, quantification, and SNP genotyping. One advantage of TaqMan probes is that there must be specific binding between the template and probe to product a fluorescent signal.
HybProbe probes are real-time PCR probes designed for the Roche LightCycler PCR System. These probes work by bringing together two separate labeled oligonucleotide probes, one with a donor and one with an acceptor fluorophor. When both the oligonucleotides are bound to the template, the fluorescence resonance energy transfer (FRET) occurs and detected. With HybProbes, the fluorescence is measured after the annealing step, but before elongation.
SimpleProbe probes are a unique kind of real-time PCR hybridization probe where each assay only needs one probe. When the probe is bound to the target sequence, it produces a greater fluorescence then when it is not hybridized with its target based on the hybridization status. These probes are great for SNP genotyping and mutation detection, though they can also be used as standard real-time PCR primers.
Molecular Beacon probes are real-time PCR probes that produce fluorescence when bound to the target sequence. When not bound to a DNA or RNA target, the probe forms a hairpin structure where a quencher moiety is close to a fluorescent dye moiety limiting the fluorescence. When bound to template, the oligonucleotide separates allowing the dye to emit fluorescence. These probes can be used for SNP detection, real-time PCR quantification, multiplex PCR assays, and detection assays.
There are many other types of probe chemistries in addition to these four. Fluoresentric can design your assay for SYBR Green, LC and Eva Green, Scorpion probes, and more.