How KASP genotyping works

Scientists around the world have been using KASP™ genotyping assays to transform commercial agriculture for more than two decades, making food production more sustainable and improving crop yields. This proprietary technology enables fast and reliable genotyping of large to small samples of any species. 

KASP genotyping assays are based on competitive allele-specific PCR and enable bi-allelic scoring of single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels) at specific loci. KASP has been cited in thousands of peer reviewed scientific publications for hundreds of species with millions of KASP markers identified.

KASP assay mix

KASP Assay Mix contains two different allele-specific competing forward primers with unique tail sequences and one common reverse primer.

Denatured template and annealing of primer(s)

In the first round of PCR, the allele-specific primer (or primers if the sample is heterozygous) that matches the target sequence amplifies the target region in conjunction with the common reverse primer. For this illustration, the allele-Y primer binds and is extended. 

In additional rounds of PCR, levels of allele-specific tail sequence increase. The fluorophore-labelled part of the FRET cassette is complementary to new tail sequences and binds to these sequences, releasing the fluorophore from the quencher to generate a fluorescent signal. In this example, allele-Y (HEX) signal is generated and the fluorophore for the non-incorporated X-allele (FAM) remains quenched.

Bi-allelic discrimination is achieved through the competitive binding of the two allele-specific forward primers. If the genotype at a given SNP is homozygous, only one of the two possible fluorescent signals will be generated. If the genotype is heterozygous, a mixed fluorescent signal will be generated.