The internet and affordable airfare have made the world a considerably smaller place. This trend is reflected in how we study diseases and science in general. International collaborations and worldwide consortia have been established to further scientific knowledge (and not just for the free trips!).

This shift towards greater collaborative efforts is particularly evident in the study of genetic diversity and pharmacogenomics. Examining single nucleotide polymorphisms (SNPs), in conjunction with genome-wide association studies (GWAS), has opened the door to large-scale genotyping of certain patient cohorts to determine if genetic variation contributes to disease pathology or response to drugs. Often, thousands of patients make up these cohorts and their genomes are examined to find a particular set of SNPs that are particular to just those patients with the disease, or to determine if the SNPs affect particular treatments, for example responsiveness to statins, a class of drugs which inhibit the uptake of lipid.

Another field in which genotyping methodology is a key application is in agricultural bioscience. For example, SNPs that relate to healthier crops or those that better resist particular disease strains can be used as markers and selected upon in strategic breeding programs. In the case of crops that are relied on for food, such as wheat, a new disease (like wheat rust) or pest can have the potential to threaten food security in an affected area. Finding a SNP or series of SNPs that associates with resistance to this disease and integrating that SNP into a breeding program can help alleviate this risk.

But how do you genotype huge groups?

Unlike the process of genotyping your mutant yeast strain, a simple PCR reaction followed by running an agarose gel simply won’t cut it when it comes to a large cohort. When you are dealing with thousands of samples per study, only high-throughput instrumentation paired with flexible genotyping chemistry can generate the data required for a statistically powerful analysis. Fortunately, there are a few options available to you that provide low-cost, high-throughput SNP determination in large sample populations:

Hydrolysis Probe-based detection method – BHQplus probes

What do you need to know about BHQplus probes?

  • Uses a combination of two allele-specific BHQplus probes and site-specific primers (forward and reverse).
  • Each BHQplus probe contains a unique fluorescent reporter paired with a BHQ® quencher dye that extinguishes fluorescence in the absence of hybridization.
  • BHQplus probes are designed with novel duplex stabilizing chemistry (modified pyrimidine residues) incorporated into the probe that allow the design of shorter probes, enhancing mismatch discrimination and making them ideal for SNP detection.
  • BHQplus SNP assays can be designed using free web-based RealTimeDesign™ software and full support design advice.
  • These assays are compatible with limited sample size (e.g., precious biopsy samples), and a broad range of instruments, including many high-throughput formats.
  • There is a wide range of reporter dye options enabling multiplex SNP assays.

How do BHQplus probes work?

The BHQplus system is a dual-labeled hydrolysis probe-based assay that utilizes four oligonucleotides per reaction: two allele-specific probes, each with a different fluorescent label representing each allele, and two unlabeled primers flanking the genomic region of the target SNP. Importantly, each probe will contain the complement sequence specific to the SNP.

The initial step of PCR introduces heat to the reaction to denature the double-stranded DNA. Then the temperature is lowered to the point where the primers can anneal to the DNA. The Taq polymerase can then extend off the primer to synthesize a new amplicon strand. As the Taq polymerase proceeds to extend the sequence of the primer, it will encounter the probe. The polymerase contains 5’exonuclease activity that will cleave the probe and separate the fluorophore and BHQ quencher. This cleavage event permanently separates them and produces a quantifiable fluorescent signal that increases proportionally with cycle number.

The key point to this reaction is that when both probes are introduced into the reaction, they will compete with each other to hybridize with the target amplicon; the SNP present in the DNA sample will determine which probe will hybridize and outcompete the other releasing signal from one dye or the other. This causes a much greater increase in the reporter that is specific to the allele present in the sample. If both alleles are present then signal from both probes are produced. This encapsulates an assay system with data output indicating the genotype of the sample material.

Universal reporter System – Kompetitive Allele Specific PCR (KASP)

What do you need to know about KASP?

  • KASP uses a universal reporter system that instead of using a probe specific to the SNP being genotyped, it relies on the inclusion of an allele-specific primers containing an adaptor sequences, or ‘tails,’ to create the reporter dye signal. It is the adaptor tail sequence that is later recognized by a fluorophore-bound oligo cassette sequence.
  • Detection is compatible with more than one system, including qPCR machines and FRET-capable plate readers. However, KASP is typically limited to two fluorophores in a single-plex assay.
  • Pre-validated KASP SNP genotyping libraries are available for a wide variety of organisms.
  • LGC Biosearch Technologies also offers a variety of KASP genotyping services where samples can be submitted for analysis and highly accurate data will be returned (ideal if you’re rushing to get preliminary data for a grant proposal).

How does KASP work?

The KASP system uses three sets of reagents in its genotyping reaction; the assay mix containing the oligonucleotides, the KASP master mix, and your DNA sample.

A few things to note:

  • The KASP master mix is the pre-prepared, optimized reagent supplied with the kit that contains typical components of a master mix but also includes the KASP reporter system.
  • The assay mix contains three unlabeled primers: a common reverse primer and two competing allele-specific forward primers to investigate the target SNP. Each allele-specific primer has a different adapter or, ‘tail,’ sequence incorporated into the full primer sequence.
  • The universal reporter system consists of two sets of duplexed oligo cassettes. The cassette contains is complementary to one of the ‘tails’ of the allele-specific primers. One strand of the cassette is labeled with a quencher and the other strand is labeled with a fluorophore (typically FAM or HEX).
  • Primers are designed using the Kraken It is possible to purchase primers either by design or on-demand; the on-demand primers are pre-optimized and validated by the company.

The KASP assay amplification reaction takes place in four steps.

Assay components are combined with the DNA sample.

  1. PCR Round 1: The allele-specific primer that is able to bind to the SNP on the DNA strand out-competes the mismatch primer, allowing the polymerase to extend the sequence. The reverse primer also hybridizes to the target region and elongates the sequence in the other direction.
  2. PCR Round 2: A complementary strand for the allele-specific tail sequence is generated, which is complementary to the oligo cassettes that have the fluorescent reporter.
  3. PCR Round 3: This is the step of signal generation. The KASP reporter, or oligo cassette, binds to the complementary allele-specific tail sequence. This separates the quencher on one strand of the oligo cassette from the fluorophore on the other strand to produce a fluorescent signal.
  4. Signal Quantification: The fluorescence produced by KASP genotyping is typically quantified at the end of the assay (end-point PCR). Successive rounds of PCR cycling amplify the amount of allele-specific tail. Thus, increasing cassette binding and the signal observed. Much like a BHQplus assay the relative proportion of signal in each channel is then quantified to determine genotype.

SNP analysis: Two great choices, BHQplus and KASP

When it comes to biallelic discrimination, both the BHQplus and KASP systems provide fast and accurate results, especially for large numbers of samples.

 

BHQplus

Comparison of KASP and BHQPlus technologies. The performance of two SNP assays targeted to the same polymorphism using the different technologies display similar results as shown in the scatterplot.

The KASP system requires an initial investment in reagents and software, but they offer support for human cohort studies, and have large pre-made and optimized genotyping libraries for a variety of species including human, mouse, and a wide array of plants.

The BHQplus system comes with free software for probe design as well as personalized design support. Probes are optimized for your specific SNP of interest, and can be customized to the fluorescent channels of your instrument. BHQplus probe chemistry can also enable multiplexed SNP detection on some instruments.

Both systems offer a great way to accurately genotype sample material for a variety of SNPs. The only factor you need to consider is which one will best suit your needs. In a shrinking world with large expansions of data, there’s no wrong answer!

References

https://www.biosearchtech.com/support/applications/genotyping-qpcr

https://www.biosearchtech.com/support/videos/bhqplus-probe-applications

http://www.lgcgroup.com/LGCGroup/media/PDFs/Products/Genotyping/KASP-brochure.pdf

Agbio based genotyping libraries available for a variety of plants including wheat, maize, lentil and more.

Examples of KASP case studies in wheat and mice.

A more thorough explanation of how the KASP system works can be found here.