Multiplex assays facilitate amplification of multiple targets in a single reaction vessel, reducing both the time and cost of experimentation. PrimerPlex is an efficient and sophisticated tool for designing oligos for multiplex assays. Oligo Design for Multiplex PCR & High Throughput SNP Genotyping and Analysis The microspheres are internally labeled with fluorescent dyes and coupled with the anti-TAG sequences.Īllele 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. The reaction conditions for this SNP genotyping assay depends upon the anti-TAG sequence that is attached to a fluorescent microsphere. Extension only occurs if the 3' end of the allele specific primer is bound to the homologous allelic sequence. In the allele specific primer extension step, polymerases extend the primer by incorporating biotin-labeled dNTPs. No labeling of primers is done to generate this fragment. The template for the ASPE reaction is the amplified PCR fragment. The capture sequence (TAG sequence) should be at the 5' end of the primer. The SNP should be present at the 3' end of the allele specific primer for accurate hybridization.Ĥ. ASPE primers should be from the same DNA strand (per target sequence) and be synthesized for all the sequence variants.ģ. Allele Specific Primer Design Guidelinesġ. This is done with the help of an appropriate capture sequence attached to the allele specific oligonucleotide.ĭesign strain differentiation Allele Specific Primer Extension (ASPE) xMAP®assays with AlleleID®. Taking advantage of the solution phase kinetics, this technique allows sequence labeled microspheres to be used for detecting new templates. The ASPE method involves two phases, an enzymatic reaction that determines the target genotype followed by a capture on solid microsphere surface for detection. 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.
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