lfe sciences
Learn how to perform KASP genotyping test using Pangu qPCR instrument in five minutes
2024-04-28
What is KASP
KASP (kompetitive allele specific PCR) is a new genotyping technology based on single nucleotide polymorphism (SNP). This technology is based on fluorescence reading after the PCR reaction. Each well uses dual-color fluorescence to detect two possible genotypes of one sample and one site. It is one of the most efficient and economical genotyping methods. Let's take a look at how to perform KASP genotyping detection using the Pangu qPCR instrument.
KASP workflow
Step 1:
Design the corresponding probes and primers. There are three primers in total, two typing upstream with fluorescent connectors and one universal downstream (Figure A below). There are four probes in total, two fluorescent probes and two quenching probes (Figure B below). The fluorescent probes and quenching probes are complementary, and the fluorescent probes are consistent with the connector sequence (tag sequence) of the specific upstream primers.
Step 2:
PCR amplification is performed through a qPCR instrument. In the first round of PCR amplification, the allele-specific primer (3' end can be paired) can identify the specific allele template and complete the allele recognition. The reverse universal primer will also bind and complete the entire PCR process. At this stage, the fluorescently labeled probe is still bound to its complementary quenching probe and no fluorescent signal is generated. Starting from the second round of PCR amplification, a template carrying a universal tag sequence appears in the product. This step completes the introduction of the universal tag sequence into the PCR product corresponding to the SNP. Subsequently, the fluorescent probe is added to the PCR product by binding to the DNA chain complementary to the tag, so it no longer binds to its complementary quenching probe, thereby generating a fluorescent signal.
How to ensure that the probe and primer can bind as expected during the PCR process?
Because the touchdown PCR program is set, the separate binding of the probe and primer can be guaranteed. When the annealing temperature is high, the primer will first bind specifically to the template because of its high Tm value, and then amplify under the action of the Taq enzyme, thereby increasing the original template for probe amplification. As the annealing temperature decreases, the probe begins to bind to the template and then produces a fluorescent signal.
Step 3:
After the Pangu qPCR test is run, obtain KASP data under the SNP analysis menu.
Pangaea real time PCR System Assists KASP Genotyping Detection
•The heating speed can reach 8.5℃/s, and 96 genotyping reactions can be completed quickly at one time and the results can be issued, with high product yield and good specificity
•The temperature control accuracy of ±0.1℃ meets the strict requirements of KASP for amplification temperature, ensuring the specificity of identifying single base differences
•Supports 12-column temperature gradient function, which is convenient for rapid optimization of the conditions required for KASP amplification
•Optical waveguide top detection, no edge effect and optical path difference, no calibration required
•6-color detection channel, supports various commonly used fluorescent markers, and is compatible with commonly used KASP typing kits
KASP application
KASP technology is flexible, cheap, and accurate. Compared with other technologies, it has certain flexibility and is easier to achieve high-throughput and automated detection. In addition, KASP uses a universal probe that can be used in conjunction with a variety of different gene-specific primers without the need to synthesize probes for each specific site, reducing the reagent cost of the experiment. This technology has been widely used in the fields of animal and plant breeding, germplasm resource identification, genetic map construction, and seed purity identification.