Allele-specific polymerase chain reaction: Mechanism, experimentation, and application

Introduction. The purpose of our research is to develop auxiliary assays to augment an extremely high-throughput SNP analysis facility. The miscellaneous nature of our assays will demand a high degree of versatility but will not require extreme high throughput application. The spectrum of our requirements therefore will span small numbers of single locus analyses to moderately large numbers of multiplexed runs. The appropriate assay format for any project is based on the number of SNP loci versus the number of individuals needed to produce meaningful data. Based on this paradigm each project can be efficiently designed and optimized with our methodology. The core of our approach is based on the mechanism of ASPCR. This has traditionally been viewed as only a moderately discriminating method of SNP analysis. We feel that recent advances described in literature will improve the specificity of the method.; Problem. Major issues facing high-throughput facilities are DNA quality, DNA quantity, and sample identity. DNA quality is important, because degraded samples may not perform as well as high molecular weight samples. Also, samples from large projects may be obtained at a number of collection sites, processed by a variety of methods and stored under different conditions. Sample identity can be confused due to clerical errors during the collection or processing phases. While all samples are quantitated by picogreen or UV absorbance before processing this cannot resolve sample quality or misidentification. Furthermore, the addition of one or more polymorphic sites to a validated multiplex assay is usually cost prohibitive. However, the desire to do so is not uncommon. We wished to develop an inexpensive auxiliary assay to augment data production for particular projects.; Solution. We implemented Allele-Specific PCR as the base methodology to assess the quality of samples entering the analytical queue of the Illumina(TM) platform. Optimization of reaction conditions, and thermal cycling profiles assisted in the further development of the method. These standard conditions were used to develop a more sophisticated assay for single locus assessment. Optimization of this procedure led to a novel method to perfect ASPCR selectivity.