What is PACE genotyping?
PACE™ (PCR Allele Competitive Extension) genotyping is a fluorescent, competitive allele-specific PCR genotyping technology. It is ideal for biallelic discrimination of single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels)
at specific loci.
Who should use it?
The PACE genotyping system has been developed for genotyping at any scale. It also allows you to increase throughput without compromising data quality. Thus this methodology can be of particular benefit to researchers performing high volume screening
experiments, with large sample sets, such as in plant breeding projects.
How does it work?
PACE genotyping chemistry has 2 parts:
- Assay Mix—comprised of 2 allele-specific forward primers, one common reverse primer
- 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.