Stephen Little

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.

The quality of DNA extracted from liquid or dried blood is not adversely affected by storage at 4°C for up to 24 h

BACKGROUND A consistent and stable source of DNA is an essential requirement for many Biobanks. An important pre-analytical variable is the delay between sample collection and sample processing. METHODS Fresh blood samples (n = 80) were collected and either processed immediately or after storage for 24 h. The samples were either stored as liquid blood at 4 degrees C or as dried blood spots at ambient temperature on three types of paper-based storage media. The quality of the DNA extracted from the samples was measured. RESULTS No difference was observed between fresh and stored blood samples. CONCLUSIONS The quality of DNA extracted from liquid or dried blood is not adversely affected by storage at 4 degrees C for up to 24 h.

Amplification refractory mutation system linear extension: a novel, gel-free, enzyme-linked immunoassay method for DNA genotyping.

A single synthesis cycle of the amplification refractory mutation system (ARMS) was applied to the analysis of K-ras alleles amplified by polymerase chain reaction and immobilized in streptavidin-coated microtiter plates. The ARMS cycle provided the specificity and molecular switch characteristics of a conventional ARMS assay. This allowed linear extension from an allele-specific primer and the incorporation of digoxigenin-labeled deoxyuridine monophosphate from digoxigenin-11-deoxyuridine triphosphate in the presence of the appropriate K-ras allele. Any digoxigenin-labeled deoxyuridine monophosphate substitution was then demonstrated by enzyme-linked immunoassay with a colorimetric endpoint. This method is capable of detecting underrepresented acquired mutations, and this has been shown by the unambiguous detection of specific K-ras mutations in cell line DNA/normal human genomic DNA admixtures. The characterization of K-ras mutations in frozen colorectal tumor samples and histologic material is also described.