Probe-based qPCR Approaches for Detection of SNPs and Validation of Molecular Diagnostics  |  October 30, 2014

Additional Resources:

• SNP Genotyping with Real-Time PCR (qPCR) Assays
• BHQ Dye Selection Chart
• RealTimeDesign™ Software
• Ordering BHQplus Probes

Part 1: A Multiplex Two-Color Real-Time PCR Method for Quality-controlled Molecular Diagnostic Testing of FFPE Samples

Professor Willey’s team at the University of Toledo Health Sciences Campus in Ohio has developed a multiplexed method for two-color fluorometric, real-time, reverse transcriptase qPCR from formalin-fixed, paraffin-embedded fine needle aspirate biopsies. The assay, published in PLoS One in February, 2014 uses a patented internal standards mixture, as well as external standards and a pre-amplification step, to enable measurement of transcription levels of three lung cancer-associated genes and a control gene. These genes comprise a previously reported lung cancer diagnostic test.

The fluorometric probes used in the method are labeled with Black Hole Quencher Dyes in BHQplus Probes produced by LGC Biosearch Technologies. Key elements of the group's approach – multiplex PCR with an internal standards mixture (ISM), and the use of two rounds of PCR – received a US patent in 2009. The new method builds on similar two-color fluorometric technology for HIV testing, such as that used by COBAS, but includes internal controls not just for genes of interest but also for loading control genes. Thus, the COBAS HIV test is a single analyte measured against a single internal standard, using two probes with two different signal colors to sort them out during the PCR process.

In contrast, the method developed by Willey’s group includes a mixture of internal standards for multiple targets, both loading control and target genes, and a sequence-specific probe with one fluorometric signal color for each native template and a sequence-specific probe with the second fluorometric signal color for the internal standard for each target gene measured. The BHQplus Probes were optimal for this application because shorter probe sequence length is required to ensure specific binding to each targeted site.

Speaker: James C. Willey, George Isaac Professor for Cancer Research, University of Toledo Health Sciences Campus, Consultant for Accugenomics, Inc.

Download presentation slides provided by James C. Willey

Part 2: BHQplus Probes and the Array Tape™ Platform: A Successful Combination for Accurate and Economical SNP Genotyping

Single Nucleotide Polymorphisms, or SNPs, are small genetic variants found throughout the genomes of plants and animals. SNPs are studied for a wide variety of reasons, ranging from clinical diagnostics to maker-assisted selection in agricultural breeding programs. Recent improvements in genomic sequencing technologies have increased our knowledge and understanding of SNPs, and the utilization of SNPs in genetic studies has increased accordingly. This has created a need for laboratory instrumentation and reagents that enable accurate and cost-effective analysis of SNP genotypes.

Allele-specific hydrolysis probes are a common choice for PCR-based SNP analysis because the assays are well understood, easily automated, and compatible with many commercially available master mixes. BHQplus probes and SNP genotyping assays from Biosearch Technologies are industry-leading in quality, specificity, and economic value. They are compatible with real-time and endpoint PCR platforms, including the Array Tape Platform from Douglas Scientific (now part of Biosearch Technologies). The Array Tape Platform is an open and customisable laboratory automation solution optimized for high-throughput SNP genotyping with miniaturized reaction volumes.

Here we will discuss the use of BHQplus Probes and SNP genotyping assays in combination with the Array Tape Platform. Using examples from clinical diagnostics, animal research, and plant research, we will demonstrate SNP genotyping assay design and optimization, and show that BHQplus Probes in combination with the Array Tape Platform provide accurate, reliable SNP genotyping results while producing significant savings on a cost per data point basis.

Speaker: Cassie Keppel, Laboratory Operations Manager, Douglas Scientific (now part of Biosearch Technologies)

Download presentation slides provided by Cassie Keppel