Allele Specific Primer Extension (ASPE) is a solution based, sequence specific enzymatic reaction technology that can be used to assay multiple SNPs in a single tube. The ASPE method involves two phases, an enzymatic reaction that determines the target genotype followed by a capture on solid microsphere surface for detection. Taking advantage of the solution phase kinetics, this technique allows sequence labeled microspheres to be used for detecting new templates. This is done with the help of an appropriate capture sequence attached to the allele specific oligonucleotide.
Design strain differentiation Allele Specific Primer Extension (ASPE) xMAP®assays with AlleleID®.
The template for the ASPE reaction is the amplified PCR fragment. No labeling of primers is done to generate this fragment. In the allele specific primer extension step, polymerases extend the primer by incorporating biotin-labeled dNTPs. Extension only occurs if the 3' end of the allele specific primer is bound to the homologous allelic sequence.
The reaction conditions for this SNP genotyping assay depends upon the anti-TAG sequence that is attached to a fluorescent microsphere. The microspheres are internally labeled with fluorescent dyes and coupled with the anti-TAG sequences.
Allele specific oligonucleotides can be developed by following the assay design guidelines outlined here or by using PrimerPlex, a software tool for designing ASPE assays for multiplex analysis on xMAP based systems.
PrimerPlex is an efficient and sophisticated tool for designing oligos for multiplex assays. Multiplex assays facilitate amplification of multiple targets in a single reaction vessel, reducing both the time and cost of experimentation.
Primer design for multiplex PCR presents several challenges which include primer dimers, inability to separate amplicons with similar electrophoretic mobility and mis-priming due to nonspecific binding to non-target DNA templates. PrimerPlex uses proprietary algorithms to design optimal multiplex primer sets under uniform reaction conditions for over 100 sequences. The primer sets are identified after screening all the primers in a pool and minimizing Tm mismatches to ensure specific amplification and high signal strength. In this process, it analyzes millions of possible multiplex primer sets in a few seconds and presents a list of alternate sets. You can specify minimum product size differences amongst the set of designed primer pairs for better visualization of bands on a gel. To assure primer specificity, primers can be BLAST searched from PrimerPlex against any of the genomic databases available at NCBI.