DT has partnered with 3CR Bioscience (UK) to provide a cost-effective, PCR-based, SNP and indel genotyping system—PACE, or PCR Allele Competitive Extension, genotyping for screening projects. Free, optimized primer design is provided. The primers are subsequently synthesized by IDT, the global leader in oligonucleotide synthesis. 3CR Bioscience provides their high-performing PACE Genotyping Master Mix. Together these reagents yield cost effective, optimally designed genotyping assays that allow increased throughput without compromising data quality.
Read more details about our partnership.
PACE genotyping chemistry has 2 parts:
- 1. Assay Mix—comprised of 2 allele-specific forward primers, one common reverse primer
- 2. Master Mix—containing all of the components required for PCR and generation of fluorescent signal
Each forward primer contains a unique tail sequence that corresponds with 1 of 2 universal FRET (fluorescence resonant energy transfer) cassettes in the master mix; one labelled with FAM™ dye and the other with HEX™ dye. During thermal cycling, the relevant allele-specific primer binds to the template and elongates, thus attaching its tail sequence to the newly synthesized strand. The complement of the allele-specific tail sequence is generated during subsequent rounds of PCR, enabling the FRET cassette to bind to the DNA. Through binding, the dye within the FRET cassette is no longer quenched and emits fluorescence.
If the genotype at a given SNP is homozygous, only 1 of the 2 possible fluorescent signals will be generated. If the genotype is heterozygous, both fluorescent signals will be generated.
The universal PACE Genotyping Master Mix is optimized to provide improved reduction in non-specific amplification compared to other competitive allele-specific PCR master mixes. Select PACE Genotyping Master Mix without ROX normalizing dye, or with high ROX (500 nM), standard ROX (150 nM), or low ROX (25 nM). When working with crude samples that contain large amounts of PCR inhibitors, the PACE-IR™ Genotyping Master Mix, which counteracts these inhibitors, is also available with and without ROX dye.