SNP Genotyping

Single nucleotide polymorphisms (SNPs) are the most common form of mutation in the genome. MDS can identify candidate SNPs in mRNA transcription regions (cSNPs) or in nearby genomic regions by sequencing. For genes specifically associated with traits of interest, the SNP targeted may be the cause of the variant trait.

Individual SNP Profiling:

SNP profiling services are offered for pharmacokinetic, pharmacodynamic, safety, tolerability, and efficiency outcomes through genotype/phenotype correlation analysis. To support these SNP profiling applications, MDS offers PCR/gel-based analysis (i.e. RFLP assays) and other genotyping services for a number of drug metabolism enzymes and other clinically relevant genes:

5HHT Alpha 2 Macroglobulin (A2M) APOE COMT CYP2A6 CYP2C19

CYP2C9 CYP2D6 CYP3A4 CYP3A5 DRD2 F2 Factor V Leiden

FLT3 MDR1 NAT2 PPAR (alpha, gamma, gamma2) SDC4 UGT1A1

Custom assay design of customer specified SNPs can be performed from either text based sequence files containing the polymorphism and surrounding sequence or from SNP accession number information.

We will design oligonucleotide sequences for PCR amplification of the genomic region surrounding the SNP locus and the detection oligonucleotide that hybridizes adjacent to the SNP locus.

Pre-Production Optimization

Before genotyping is scaled up for a particular SNP, several optimization and standardization reactions need to be performed. These include:

• Selected florescent dye (such as SYBR green) needs to be added in order to detect the resulting PCR products. The levels of the dyes need to be adjusted to provide a stable background with a sufficient signal to background ratio. The ABI SYBR green kit provides a good system. .
• A set of reactions will have to be set up to verify the region of genomic DNA containing the SNP is amplifiable and that allelic discrimination is achievable
• The TaqMan technology allows one to perform the PCR reaction in a 20 ul volume. The actual useable reaction volume needs to be determined.

Primer Design

• Automated primer design using ABI software.
• Design two sets of primers for each SNP. One will be ordered but a second set will be pre-designed in case of a failure.
• Primers will be standardized to be between 18-22 mers with a constant Tm, GC ratio, etc., in order to favor success of amplification under a single set of PCR conditions.
• Expected PCR products will fall into the range of 100-250 bp but may be longer if necessary.