David Whitcombe

Detection of PCR products using self-probing amplicons and fluorescence

Molecular diagnostics is progressing from low-throughput, heterogeneous, mostly manual technologies to higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays, and a number of techniques rely on energy transfer between a fluorophore and a proximal quencher molecule. In these methods, dual-labeled probes hybridize to an amplicon and changes in the quenching of the fluorophore are detected. We describe a new technology that is simple to use, gives highly specific information, and avoids the major difficulties of the alternative methods. It uses a primer with an integral tail that is used to probe an extension product of the primer. The probing of a target sequence is thereby converted into a unimolecular event, which has substantial benefits in terms of kinetics, thermodynamics, assay design, and probe reliability.

Multiplexed assays for detection of mutations in PIK3CA.

BACKGROUND Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. METHODS We combined Amplification Refractory Mutation System (ARMS; AstraZeneca) allele-specific PCR and Scorpions (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). RESULTS These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. CONCLUSIONS Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.

Advances in approaches to DNA-based diagnostics

The most tangible advances in DNA diagnostics during the past year have been in enhancing existing techniques to simplify their use and improve throughput. This has led to simplified genotyping methods using homogeneous analysis coupled with spectral data output. Miniaturisation and increased throughput have also been achieved through improvements in DNA chip technology.

Design and Use of Scorpions Fluorescent Signaling Molecules

A number of probe systems exist for the real-time detection of PCR products. Scorpions are a unique method wherein primer and probe are combined in a single oligonucleotide. During the PCR, the probe element becomes linked directly to its complementary target site with beneficial consequences. In particular, the unimolecular mechanism of probe/target hybridization ensures rapid, reliable, and robust probing of a chosen amplicon. We discuss the design and use of Scorpions and compare their use with similar systems.