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SNP Analysis

AB Allelic Descrimination

Description
AB Taqman SNP Genotyping consists of single assays that contain two differently labeled probes for each allele of the locus. Typically, each assay is run individually with limited multiplexing ability. Among the SNP platforms available at the Core, this one is most cost-effective for projects that are interrogating a small number of SNPs (up to 10) and 100 or fewer samples. The benefit of using these assays is that they are pre-validated and require minimal optimization.

Science
This method requires two differently labeled TaqMan probes (either standard dual-labeled probes or MGB probes) in which the polymorphic base of query is designed in the middle third of the probe and a set of primers. Each allele-specific probe has essentially the same sequence (with some exceptions) but is labeled with either a FAM or VIC reporter molecule. A PCR reaction for the locus of interest is performed on standard PCR machines with TaqMan probes for both alleles in the reaction mix.

Protocol
Please consult the Applied Biosystems manual for more information.

Pricing
Please contact the Core for more information.

What to Provide
Please contact the Core for more information.

Sequenom SNP Analysis

Description
Sequenom SNP Genotyping is a mid- to high-throughput method that uses multiplex PCR coupled with mass spectrometry to interrogate up to 40 SNPs per reaction. Among the SNP platforms available at the Core this method is more cost-effective for larger studies (greater than 10 SNPs, 100 samples). It is a good platform for sub-whole genome study applications and is widely used for fine mapping and validation studies and for routine applications that employ fixed SNP panels. The increased sensitivity of mass spectrometry over PCR-only methods makes the platform particularly sensitive and quantitative for low abundant mutations. The validation of assays requires extra time allocated before starting the project.

Science
The Sequenom SNP method (called iPlex Gold Genotyping) is based on multiplex PCR followed by a single base primer extension reaction. After the PCR, the remaining nucleotides are deactivated by SAP treatment. The single base primer extension step is performed, and the primer extension products analyzed using MALDI TOF mass spectrometry (Matrix-Assisted Laser Desorption/Ionization - Time of Flight).

Protocol
Temporary protocol available here.
Also see Sequenom protocol.

Pricing
Please contact the Core for a quote. Please provide the number of SNPs and the number of samples. The number of samples should be in multiples of 96, which best utilizes the lay-out of the platform.

What to Provide
Please provide the list of SNPs with their “rs” ID numbers to design multiplex assays. High quality DNA (260/280 ratio > 1.7) is required at 15 +/- 5 ng/ul concentration in 96-well plates. The total quantity of DNA depends on the total number of reactions required to run the requested SNPs.

Additional Resources
A searchable database of core facilities at all UCSF campus locations, provided by the Clinical and Translational Science Institute at UCSF, is available here.