SNPSCALEProtocol.docVIP

SNP SCALE Protocol. Overview SNP SCALE (SNP Scoring by Colour And Length Exclusion) is a SNP genotyping system based on allele specific PCR. Allele specific PCR relies on differences in the binding efficiencies of a perfect match versus a mismatch for each of the allele specific forward primers to distinguish between the two SNP alleles. The advantages of allele specific PCR include all of the normal benefits associated with PCR: high sensitivity, repeatability, can work on small amounts of tissue and degraded tissue, rapid, and flexible. However there are some problems unique to allele specific PCR for SNP genotyping, in particular the cost of fluorescent primers and potential errors associated with non-specific primer binding. SNP SCALE has been designed to take advantage of the benefits of allele specific PCR, while trying to overcome some of the drawbacks. SNP SCALE uses a specially modified oligonucleotide, Locked Nucleic Acids (LNAs), in the 3’ SNP position to overcome the problems of non-specific binding, and it uses 5’ tailing and universal fluorescent oligos (UFOs) to overcome the problems of fluorescent labelling costs. The 5’ tails, which are slightly different in length, have the added advantage of facilitating allele scoring by both colour and length exclusion. Described here is the SNP SCALE protocol, including PCR set-up and reaction conditions. Also discussed are issues associated with primer design. Because there are five primers in the reaction mixture, there are 25 possible forward and reverse primer combinations, exponentially increasing the chance of non-specific binding. Thus good primer design is critical. LNAs (Locked Nucleic Acids) LNAs are a specially modified nucleic acid that increase primer binding specificity, and thus decrease mis-priming. Also they raise the effective annealing temperature of the primer by about three degrees, so that the effective difference in annealing temperature between the allele specific primers is further incr