SNP Genotyping*

SNP Genotyping
Single nucleotide polymorphism (SNP) genotyping has become central to the advancement of molecular genetics. At the Molecular Diagnosis and Genotyping Facility, we use the ABI Prism® 7900HT allelic discrimination platform.

SNP Genotyping Method

The primary approach for SNP genotyping is the use of the ABI SNP genotyping assay with readout of the genotypes using the ABI 7900HT instrument (Applied Biosystems, Inc., Foster City, CA). The method combines PCR amplification and detection in the same reaction. The method is based on the 5' nuclease activity of Taq DNA polymerase. A PCR is performed using primers that will amplify the DNA region containing the SNP of interest. Included in the reaction are two allele-specific fluorogenic probes, each consisting of a different fluorescent reporter dye and a fluorescent quencher. In the intact probe, the proximity of the quencher to the fluorphore causes fluorescence resonance energy transfer (FRET), reducing the fluorescence from the reporter dye. During PCR, the 5' nuclease activity of Taq digests the allele-specific probe bound to the region of the SNP, releasing the fluorescent dye from the quencher and allowing generation of a fluorescence signal. Depending on which dye signal is generated, the SNP alleles are determined. If only one dye signal is detected, the SNP is homozygous for the allele corresponding to the allele-specific probe, and if both dyes are detected, then the SNP is heterozygous.

The ABI reactions contain 5 ng of genomic DNA, specific PCR primers, two allele-specific probes, dNTPs, Taq DNA polymerase and buffer according to the manufacturer's specifications. Reactions are set up in 384-well plates using a Biomek FX (Beckman Coulter), and cycled in MJ Research 384 well thermal cyclers. SNP alleles are called using ABI software that provides scatter plots of allelic calls.

Using the SNP Genotyping Service

1. The investigator identifies SNPs of interest and orders the primer/probe sets from ABI at preferred UPenn pricing (see below for prices).

Finding SNP database ID numbers

Instructions for designing assays, submitting sequences to Applied Biosystems for design, and purchasing the assays

Instructions for the submission of ABI Primer and Probes to the Cancer Center

2. The investigator prepares experimental DNAs in specific 96-well plates at the specified concentration and format.

Submitting DNA to the Facility

3. The investigator brings the primer/probe sets and DNA to the Molecular Diagnostic and Genotyping Facility on 7 West Gates at the Hospital of the University of Pennsylvania. The completed SNP submission Form and Specimen Identification Files should be emailed to Kathakail Addya, Technical Manager of Molecular Diagnostic & Genotyping Facility.

Specimen Identification Form

4. The MDGF staff will test the SNP reagents on controls and report back to the investigator. If the SNP reagents work well, and with the approval of the investigator, experimental DNAs will be tested for all SNPs.

5. Results are reported to the investigator in several formats.

Example of SNP Results

Controls

All SNP reagents are tested on a set of control DNAs to assess the quality of the assay and the heterozygosity of the SNP. Controls include 40 African-American, 40 Caucasian and 1 CEPH family DNAs. Results of controls are reported to the investigator to determine if the experimental specimens should be tested.

Information on Human Variation Controls for SNP Genotyping

*Support for this project was awarded by the Abramson Cancer Center of the University of Pennsylvania, and is funded, in part under a grant with the Pennsylvania Department of Health. The Department specifically disclaims responsibility for any analyses, interpretations or conclusions.

To obtain more information on the SNP genotyping service, please contact the facility manager, Dr. Kathakali Addya via email or by calling 215-662-6589, or the acting facility director Dr. Don Baldwin via email, or by calling 215-662-6550.