Single Nucleotide Polymorphisms (SNPs) have been widely studied for over 15 years, and have applications in forensics, disease susceptibility studies, pharmacogenomic and other personalized medicine applications, crop and livestock breeding programs. Numerous genetic methods and companion platforms have been developed for SNP detection encompassing DNA sequencing, microarrays, mass spec, and PCR based techniques. For high sample throughput applications, PCR analysis has emerged as the method of choice for rapid targeted SNP analysis, facilitated by new platforms and reagent cost improvements. On October 30, 2014, Biosearch Technologies sponsored a webinar showcasing the utility of the BHQplus® probe technology for qPCR based SNP analysis. Presenter Cassie Keppel, Laboratory Operations Manager at Douglas Scientific, shared some compelling data showing the accurate and economical utility behind the use of Biosearch’s BHQplus probes on Douglas Scientific’s Array Tape® platform. In this article, we highlight some of Cassie’s findings on the dramatic cost-savings that the BHQplus probe and Array Tape platform combination offers.
The Douglas Scientific Array Tape platform serves as a microplate replacement in a highly automated platform workflow that incorporates liquid handling, thermal cycling and fluorescence detection. Data generated from the Array Tape smaller 1.6 μL reaction volumes compare favorably with standard reaction volumes from traditional real-time PCR instruments. Data was presented for several SNP genotyping experiments with Human, Corn, and Bovine sources. BHQplus probes form highly stable duplexes with DNA targets, and allow for shorter probe design with enhanced target specificity. These characteristics allow for excellent sequence discrimination enabling sensitive end-point SNP calling in the qPCR assay.
Human, Corn and Bovine SNP Studies
The human SNP assays that Cassie presented were chosen for their availability as widely-recognized, pre-designed, commercial assays. The commercial assays were compared to assays designed using off-the-shelf master mixes with BHQplus probes. All studies used BHQplus probes designed with Biosearch Technologies’ RealTimeDesign™ software. Corn and Bovine SNP studies used master mixes purchased from a variety of manufacturers. The instrument platforms used were the ViiA7 with 384 well plates, and the Douglas Scientific Array Tape platform with 384 well arrays. Examples of all SNP genotyping assay results were reported using cluster analysis plots.
Factors Influencing Assay Development
Multiple considerations factor into the choice of platform and reagents for probe-based SNP genotyping. The cost of each data point must account for the quality of sample preparation, hands on preparation time, the cost of the platform and daily operation, and the compatibility between reagents such as the particular assay format and the master mix. While pre-designed assays for certain organisms are available for purchase on various platforms by those who are not interested in high throughput analysis, their high cost can limit the economics of their use in high throughput. Furthermore, interrogation of novel SNP sites or samples from uncommon species further limits the utility of “off-the-shelf” assays.” Not so with BHQplus assays designed with RealTimeDesign, which offer greater affordability than many "off-the-shelf" assays that are within easy reach of those running smaller numbers of SNP samples. If you are interested in developing a high throughput SNP assay that makes the best use of today's assay economics, the Array Tape platform deserves special consideration when specifying an assay platform of choice, and the BHQplus probe technology provides cost savings with multiple platforms.
Ultimately, platform-dependent reaction volume determines the cost of the master mix and assay components per data point for less expensive high throughput PCR platforms, and the most economical methods for high volume sample testing are shrinking reaction scales to microfluidic SNP assays (see table below).
Table of the comparative percent cost per reaction across platforms, master mixes, and assay formats.
*Table borrowed from C.Keppel’s (Douglas Scientific) presentation in the webinar.
The Bottom Line
BHQplus probes were found to be both effective and accurate in a wide variety of samples encompassing human and Ag/Vet applications. The Array Tape Platform enables miniaturization and automated PCR-based analyses without sacrificing accuracy or call rates.
Master mix and assay cost considerations are summarized in the table above. For high throughput customers, the savings afforded using the Array Tape platform with BHQplus probes are significant and compelling reasons for any customer contemplating SNP assay development.
For more information on BHQplus probes, please visit https://www.biosearchtech.com/bhqplus
RealTimeDesign Software can be found at: https://www.biosearchtech.com/realtimedesign
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