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TaqMan SNP Genotyping Assays provide optimized assays for genotyping single
nucleotide polymorphisms (SNPs). These products use the 5' nuclease
assay for amplifying and detecting specific SNP alleles in purified genomic
DNA samples. Each assay allows researchers to genotype individuals for
a specific SNP.
Assay Components:
Each SNP Genotyping Assay Mix contains:
- Sequence-specific forward and reverse
primers to amplify the SNP of interest
- Two TaqMan MGB probes: one probe
labeled with VIC dye detects Allele 1 sequence, one labeled with FAM dye
detects Allele 2 sequence.
- A minor groove binder (MGB). This
modification increases the melting temperature without increasing probe
length which allows for shorter probes. This results in greater
differences in melting temperature values between matched and mismatched
probes, with produces more allelic discrimination.
- A nonfluorescent quencer (NFQ) at the 3'
end of the probe. Because the quencher does not fluoresce, ABI
sequence detections systems can measure reporter dye contributions more
accurately.
5' Nuclease Assay:
During PCR, the following events occur:
- Each TaqMan MGB probe anneals
specifically to a complementary sequence between the forward and reverse
primer sites. When the probe is intact, the proximity of the
reporter dye to the quencher dye results in suppression of the reporter
fluorescence primarily by Forster-type energy transfer.
- AmpliTaq Gold DNA polymerase cleaves
only probes that are hybridized to the target. Mismatches between a
probe and target reduce the efficiency of probe hybridization.
Furthermore, the enzyme is more likely to displace a mismatched
probe without cleaving it, which does not produce a fluorescent signal.
- Cleavage separates the reporter dye from
the quencher dye, which results in increased fluorescence by the reporter.
The increase in fluorescence signal occurs only if the amplified target
sequence is complementary to the probe. Thus, the fluorescence
signal generated by PCR amplification indicates which alleles are present
in the sample.
ABI Genotyping Workflow:
Services Offered by us: In addition
to offering full SNP assay service we also offer services at multiple levels
to accommodate researchers needs.
Full service: We setup the plate,
run the PCR and analyze the data. The cost for this service is $5.00/well
Partial services: Researchers
could set-up 384 well plates in their own lab and then bring the plate
to the facility to run the PCR. The cost for this service is $60.00/plate.
Alternatively, the setup and PCR could be carried out in researcher's lab and then
simply bring the pate to the resource to post read using 7900. The fee for
this service is $30.00/plate.
If using partial services the 7900 must
be reserved by the researcher at :
ABI Genotyping Catalog Assays:
To browse the Appiled Biosystems catalog of TaqMan SNP Genotyping assays,
you will need a user name and password for login at this site:
ABI Store Login. The cost is usually $150-200 per assay.
Each assay has approximately 750 reactions (384-well plate format).
Additionally, TaqMan Universal PCR Master Mix is required to perform the
assay.
TaqMan SNP Genotyping Assays provide the largest collection of ready-to-use
SNP assays available. All assays were designed using powerful
bioinformatics pipelines and software, as well as genomic information from
Celera Genomics and public databases:
- TaqMan Validated SNP Genotyping Assays:
>150,000 gene-centric assays. These are the highest performing SNP
genotyping assays, which have undergone minor allele frequency (MAF)
validation on 2 or 4 ethnic group populations (45 individual samples per
ethnic group).
- TaqMan Coding SNP Genotyping Assays:
~30,000 assays for the detection of informative and putative functional
SNPs in gene-coding regions.
- TaqMan Pre-Designed SNP Genotyping
Assays: ~1.7 million genome-wide and genome-unique in silico
assays. These are manufactured and functionally tested at ABI upon
ordering.
If no primer assays can be found for the
your gene(s) of interest, primers can be designed using Primer Express
on the facility workstation.
The information above was adapted from the
TaqMan SNP Genotyping Assays Protocol (#4332856B)found
at the ABI website. |
