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.