Molecular Assays Developed to Detect Genetic Mutations

Several new assays have been developed to detect genetic mutations using coamplification at lower denaturing temperature polymerase chain reaction (COLD-PCR) technology.

The technology was focused on the Epidermal Growth Factor Receptor (EGFR) gene as EGFR mutations are found in a number of cancers, including lung and colorectal cancers. The enhanced analytic sensitivity of COLD-PCR will allow clinicians to use smaller amounts of sample for their genetic analyses and less intrusive sample collection methods such as a blood draw for serum or plasma preparation, a urine catch, fine needle aspirate or bronchial lavage for genetic analyses.

COLD-PCR was developed by Transgenomic Inc., (Omaha, NE, USA) who conducted a study of the ability of COLD-PCR to detect EGFR mutations in exons 19-21. Study results indicate that the COLD-PCR assays detect all exon 19 deletions, the T790M mutation in exon 20 and all mutations in exon 21 in the specific regions amplified, including L858R and L861Q point mutations. The sensitivity of detection when confirmed using Sanger sequencing is 0.05% to 0.2% using plasmid and cell line DNA dilutions. Limit of Detection experiments have indicated that the mutant DNA can be detected in a plasma/serum sample when as little as five to 10 copies of mutant DNA are present.

This genetic testing approach that selectively amplifies mutant DNA by recognizing that mutant DNA strands denature at lower temperatures in a PCR reaction compared with normal mutant-free DNA. Specific enhancements of the COLD-PCR methodology enable the detection of all of the mutations present in the region amplified. Therefore, prior knowledge of specific mutations is not necessary. Using this patented technique, COLD-PCR amplifies mutant DNA with minimal amplification of the normal DNA.

Craig Tuttle, M.S., M.B.A., CEO of Transgenomic, commented, "One of our key corporate strategic initiatives is to develop ultra-high sensitivity assays for the detection of low-level DNA mutations in body fluids. We believe COLD-PCR provides us with the analytical sensitivity and accuracy to achieve this goal."

The study was conducted in collaboration with the Dana-Farber Cancer Institute, (Boston, MA, USA).

Related Links:
Transgenomic Inc.
Dana-Farber Cancer Institute