Kompetitive Allele Specific PCR (KASP) is a homogeneous, fluorescence‑based genotyping technology that enables bi‑allelic discrimination of single‑nucleotide polymorphisms (SNPs), insertions/deletions (indels), and other sequence variants. The method relies on competitive binding of two allele‑specific forward primers, each bearing a unique tail sequence, together with a common reverse primer. During PCR amplification, the allele‑specific primer that matches the target DNA sequence is preferentially extended, incorporating its tail into the amplicon. Post‑amplification, the tail sequences serve as binding sites for universal fluorescence reporter cassettes that emit distinct colors (commonly FAM and HEX/VIC). The resulting fluorescence intensity for each dye allows unambiguous assignment of the genotype as homozygous for allele 1, homozygous for allele 2, or heterozygous.
Key components
| Component | Function |
|---|---|
| Allele‑specific forward primers | Contain a 3′‑terminal base complementary to one of the two possible alleles and a 5′ tail that is unique to each allele. |
| Common reverse primer | Binds downstream of the target SNP/indel region and participates in amplification of both allele‑specific products. |
| Fluorescent reporter cassettes | Universal primers with a fluorophore (e.g., FAM or HEX) that anneal to the tail sequences introduced by the allele‑specific primers during the read‑out stage. |
| PCR master mix | Provides DNA polymerase, dNTPs, Mg²⁺, and buffer optimized for competitive allele‑specific amplification. |
Workflow
- Assay design – For each SNP, two allele‑specific forward primers are designed, each ending with the variant base and carrying a distinct tail sequence. A common reverse primer is also designed downstream of the polymorphic site.
- PCR amplification – The reaction mixture contains the primer pair set, fluorescent reporter cassettes, and DNA template. During the early cycles, the allele‑specific primers compete for binding; only the perfectly matched primer extends efficiently.
- Fluorescence detection – After amplification, the universal reporter primers bind to the tail sequences incorporated into the amplicons. The fluorophore associated with each tail emits a signal measured by a plate reader or real‑time PCR instrument.
- Genotype calling – Data analysis software plots the fluorescence intensities (e.g., FAM vs. HEX) to generate a scatter plot where clusters correspond to the three possible genotypes.
Advantages
- High throughput – Compatible with 96‑, 384‑, and 1536‑well plate formats, enabling thousands of assays per run.
- Cost‑effective – Uses standard PCR reagents and does not require expensive probes or ligation steps.
- Flexibility – Applicable to a wide range of organisms (plants, animals, microbes) and can be used for marker‑assisted selection, population genetics, and pharmacogenomics.
- Robustness – Tolerant of modest DNA quality variations and integrates easily into existing qPCR platforms.
Limitations
- Bi‑allelic focus – Primarily designed for SNPs with two alleles; detection of multi‑allelic variants requires separate assays.
- Allele‑specific primer design – Requires careful optimization to avoid non‑specific amplification, especially in regions with high sequence similarity.
- Limited to known variants – The method is genotype‑driven; it cannot discover novel polymorphisms without prior sequence information.
Typical applications
- Marker‑assisted breeding – Rapid screening of cultivars for disease‑resistance or quality traits.
- Genome‑wide association studies (GWAS) – Validation of candidate SNPs identified in discovery phases.
- Quality control in seed or livestock production – Confirmation of cultivar or breed identity.
- Pharmacogenomics – Genotyping of drug‑response alleles in clinical research.
Commercial availability
KASP chemistry is commercialized by several biotechnology companies under proprietary names (e.g., LGC‑Biosearch Technologies’ “KASP™ Genotyping Reagents”). Kits include pre‑validated primer sets for many common SNPs, as well as services for custom assay development.
Reference implementation
A typical reaction (10 µL) might contain:
- 5 µL 2× KASP master mix
- 0.14 µL assay mix (containing the two allele‑specific forward primers and the common reverse primer)
- 2 µL DNA (10–50 ng/µL)
- Nuclease‑free water to volume
Cycling conditions (example):
- 94 °C for 15 min (initial activation)
- 10 cycles: 94 °C 20 s, 61 °C 60 s (decreasing by 0.6 °C per cycle)
- 30 cycles: 94 °C 20 s, 55 °C 60 s
Fluorescence is read after the final cycle.
References
- Semagn, K., Babu, R., Hearne, S., & Olsen, M. (2014). “Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): Overview and its applications in crop improvement.” Molecular Breeding, 33, 1–14.
- LGC Biosearch Technologies. “KASP™ Genotyping Technology – Technical Overview.” (Product documentation, accessed 2024).
This entry reflects information available in peer‑reviewed literature and manufacturer technical resources up to July 2026.
