Probe-Based qPCR Approaches for Detection of SNPs and Validation of Molecular Diagnostics

Drug Discovery & Development, Fundamental Research, Laboratory Technology, Life Sciences, Pharmaceutical,
  • Thursday, October 30, 2014

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 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.

Take away points: Increasingly, cancers are diagnosed through FFPE treated cytologic material from fine needle aspirates. Thus, having methods with the necessary sensitivity and quality control to reliably measure FFPE cytologic samples is increasingly important.

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. The Array Tape Platform is an open and customizable 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.

Take away points:

  • The Array Tape platform is a flexible solution that works with a variety of applications and with many chemistry options, including assays, master mixes, and sample types.
  • The Array Tape and SNP genotyping assays from Biosearch Technologies are a great combination, and together provide accurate, reliable results while producing significant savings on a cost per data point basis.
  • SNP genotyping with hybridization probes require two primers and two allele-specific probes, each with a different dye label. Probe hybridization is accurate and produces correct calls, but it is not 100% present or absent based on alleles.
  • Oligos and assays, including the software to generate them, differ between oligo manufacturers. They are not necessarily interchangeable, and may respond differently to PCR conditions.
  • SNP assay design is simple and convenient with Biosearch Technologies software and support.


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

James Willey is Professor of Medicine and Pathology, and George Isaac Professor for Cancer Research at the University of Toledo Health Sciences Campus, Toledo, OH, as well as Co-founder and Consultant for Accugenomics, Inc. (Wilmington, NC). Professor Willey is holder of more than 10 US patents in molecular diagnostic tests and methods. The current focus of research includes development of molecular diagnostic assays, novel quality controlled targeted next generation sequencing (NGS) methods, and novel approaches to understanding complex genetic traits. Professor Willey was the corresponding author on a recent paper describing a novel two-color fluorometric RT-qPCR method that provides better quality control for diagnostic testing . This method is licensed to Accugenomics.

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Cassie Keppel, Laboratory Operations Manager, Douglas Scientific

Cassie Keppel is the laboratory operations manager at Douglas Scientific in Alexandria, MN. She grew up in Alexandria and attended Saint Olaf College in where she majored in Chemistry. She completed her Master of Science Degree in Food Science and Human Nutrition at Iowa State University.

Upon graduation, Cassie worked in genetic research in the Department of Pediatrics at the University of Iowa. When her family moved to Saint Louis, MO she continued her career at the Washington University in Saint Louis School of Medicine. There she spent eight years working in genetics and molecular biology research in the fields of heritable bone deformities and oncology. She joined Douglas Scientific in the summer of 2012.

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Biosearch Technologies

Biosearch Technologies, Inc. is a privately held, ISO 9001:2008 and ISO 13485:2003 certified company, located in northern California. Founded in 1993, Biosearch is a leader in the design, development and manufacture of sophisticated oligonucleotide-based tools. Products include fluorophores (CAL Fluor®, Quasar® and Pulsar® dyes) and dark quenchers (Black Hole Quencher® dyes) to be included in custom-synthesized probe formats for real-time, quantitative PCR, Stellaris® RNA FISH, and other genomic-based applications.

Biosearch Technologies produces a variety of probe formats, including Dual-Labeled BHQ® Probes, BHQplus® Probes, Molecular Beacons, and Scorpions® Primers. All of these probe formats incorporate a BHQ dye as a quencher, which make them ideal for multiplexed qPCR. In addition to BHQ probes and primers, Biosearch manufactures oligos for in situ transcript detection, analyte targeted biosensors, next generation sequencing and aptamer conjugates.

In addition to basic R&D applications, Biosearch now leverages its demonstrated expertise in fully assembled RUO kits and in the cGMP manufacture of oligonucleotide components for molecular diagnostic assays. Biosearch Technologies is registered with the FDA (registration number 3003009052), and licensed by the State of California Department of Public Health (license number 72147) as a medical device manufacturer. As such, its GMP and Commercial Services manufacturing processes conform to all applicable requirements per 21 CFR Part 820 and ISO 13485:2003. Biosearch’s markets include Pharmaceutical, in vitro diagnostics, biotechnology, ag/bio, environmental and food testing, public health, and biodefense sectors.

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