Cold-PCR Enriches DNA Mutations in Samples

Cold-polymerase chain reaction (PCR) technology enriches mutations in samples where normal DNA predominates.

The technology helps detect cancer-related mutations where critical mutations are present at a very low percentage compared to normal DNA such as in blood and urine or in tissue samples that contain mostly normal cells. This would allow clinicians to use less intrusive methods for genetic analysis and allow more efficient use of tumor tissue samples. Additionally, the method could enhance the detection of the emergence of cancer drug resistance mutations, allowing early detection of relapse.

Transgenomic (Omaha, NE, USA), a biotechnology company, has licensed cold-polymerase chain reaction (PCR) from the Dana-Farber Cancer Institute (Boston, MA, USA). Eric Kaldjian, CSO at Transgenomic said, "Cold-PCR has the potential to further increase the sensitivity of Transgenomic's WAVE [denaturing high performance liquid chromatography] DHPLC and Surveyor Nuclease products for mutation detection in cancer and mitochondrial diseases. In combination, Cold-PCR and WAVE/Surveyor have the potential to detect one mutant copy of DNA out of a thousand to as many as ten thousand normal copies. This will be particularly valuable in cancer-related mutation detection of free DNA in blood and body fluids and in producing a mutation profile of primary tumors to predict resistance to targeted therapies. It could also have application in analysis of mitochondrial DNA heteroplasmies."

When mutant and reference DNA samples from the same gene are mixed and reannealed, variations between these sequences cause double-stranded DNA heteroduplexes to form. The WAVE System employs denaturing HPLC to separate these homo- and hetero-duplexes by ion-pairing reverse-phase HPLC. Alternatively, Transgenomic's Surveyor Nuclease cleaves such heteroduplexes with high specificity at sites of base mismatch or small insertions/deletions. It is a robust and reliable tool in analyzing DNA variations, especially where the mutant alleles are at a very low concentration within the sample (less than 1% of the total wild type allele concentration).

Cold-PCR protocols preferentially amplify heteroduplexes such that mutant alleles become enriched compared to normal alleles. The range of enrichment demonstrated to date varies from 3 to 100-fold, which will contribute to Transgenomic's target of achieving a 1/10,000 mutant to normal allele ratio detection in a routine, cost-effective, and high throughput protocol. This level of detection will allow straightforward tumor analysis via surrogate tissues such as blood and urine.

Transgenomic provides products and services for automated high sensitivity genetic variation and mutation analysis. The company offers systems, products, discovery and laboratory testing services to the clinical laboratory, to academic and medical research, and to pharmaceutical markets in the fields of pharmacogenomics and personalized medicine.

Related Links:
Transgenomic
Dana-Farber Cancer Institute