How does TaqMan SNP genotyping assays work?
The TaqMan® SNP genotyping technology utilizes the 5′ nuclease activity of Taq polymerase to generate a fluorescent signal during PCR. For each SNP, the assay uses two TaqMan® probes that differ in sequence only at the SNP site, with one probe complementary to the wild-type allele and the other to the variant allele.
What is TaqMan genotyping?
The 5′-nuclease allelic discrimination assay, or TaqMan assay, is a PCR-based assay for genotyping single nucleotide polymorphisms (SNPs). The region flanking the SNP is amplified in the presence of two allele-specific fluorescent probes. The probes do not fluoresce in solution because of a quencher at the 3′ end.
What is a genotyping assay?
Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual’s DNA sequence using biological assays and comparing it to another individual’s sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents.
What is a quencher in PCR?
The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source via Förster resonance energy transfer (FRET). As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals.
How many oligonucleotides a Taqman genotyping assay requires?
Steps 2–4 take place in the same 96-well plate. Second, a universal biotinylated oligonucleotide is employed for signal quantification. For a biallelic SNP the OLA assay requires two allele specific oligonucleotides and a common oligonucleotide.
What is allelic discrimination plot?
Allelic discrimination is a technique that is able to detect single base pair differences. It is used to discriminate between genotypes, mutations and polymorphisms within or between samples by comparing the fluorescence signal obtained using allele-specific, dye-labelled probes.
How does the TaqMan genotyping assay work?
Each predesigned TaqMan SNP Genotyping Assay in our collection includes two allele-specific TaqMan MGB probes containing distinct fluorescent dyes and a PCR primer pair to detect specific SNP targets. These TaqMan probe and primer sets (assays) uniquely align with the genome to provide unmatched specificity for the allele of interest.
How does custom Applied Biosystems SNP genotyping assays work?
Custom Applied Biosystems™ TaqMan™ SNP Genotyping Assays provide a highly flexible technology for detection of polymorphisms within any genome. TaqMan™ Assays have a simple workflow and provide the quickest way to generate genotyping data.
Is there a plugin for TaqMan SNP genotyping?
A number of related genotyping systems utilize the reporter-quencher technology (e.g. Invader®, Molecular Beacons®, Scorpion® and other probe technologies) and can be analyzed and visualized in the same way as TaqMan® probes using the SNP genotyping plugin.
What do you need for a SNP assay?
TaqMan SNP Genotyping Assays require only three reaction components for PCR: purified genomic DNA (1–20 ng), the assay solution, and TaqMan Genotyping Master Mix (or another compatible master mix) (sold separately).