Transgenomic has announced that it has licensed a high-sensitivity mutation detection technology called Cold-PCR from the Dana-Farber Cancer Institute (DFCI) in Boston.
This variation of the standard PCR technology enriches mutations in DNA samples and is a much more sensitive technique for finding low-level mutations in tissue and body fluids that are involved with a variety of diseases.
Cold-PCR was invented at DFCI by Dr Mike Makrigiorgos, who has demonstrated its effectiveness in enriching for mutations in cancer-related genes in samples where standard DNA sequencing is not sensitive enough to detect these very low concentration somatic DNA mutations.
The licensing terms include exclusive rights to commercialise Cold-PCR technology combined with Sanger sequencing as well as all applications for mitochondrial DNA analysis.
Cold-PCR will be applicable in the detection of cancer-related mutations where critical mutations are present at a very low percentage compared to normal DNA.
Examples would be in blood and urine or where the tissue collected contains mostly normal cells.
This would allow clinicians to use less intrusive methods for genetic analysis or allow more efficient use of tumour tissue samples.
The method could also enhance the detection of the emergence of cancer-drug resistance mutations, allowing early detection of relapse.
'Cold-PCR, we hope, will allow us to be able to screen patient blood for early detection of cancer, detect cancer drug resistance or relapse earlier as well as expand our mitochondrial DNA analysis toolbox,' said Craig Tuttle, Transgenomic chief executive.
'During our option period we tested the feasibility of Cold-PCR and developed practical laboratory improvements to the technology,' said Dr Eric Kaldjian, chief scientific officer at Transgenomic.
'We demonstrated reproducible 30-50 fold enrichment of mutant cancer gene DNA, without needing any a-priori information on the position of the mutation.
'What this means is that one mutant DNA molecule in a hundred is effectively changed to one in two.
'As a result, we expect that Cold-PCR has significant applications with standard Sanger sequencing methods.
'Combining Cold-PCR with Transgenomic's Wave DHPLC and Surveyor Nuclease products may have the potential to detect one mutant copy of DNA out of as many as a thousand normal copies and the sensitivity is likely to keep improving.
'This will be valuable in cancer-related mutation detection of free DNA in blood and bodily fluids and in producing a mutation profile of primary tumours to predict resistance to targeted therapies.
'It could also have application in analysis of mitochondrial DNA mutations, which can be present at very low levels,' he added.
When mutant and reference DNA samples from the same gene are mixed and re-annealed, variations between these sequences cause double-stranded DNA hetero-duplexes to form.
The Wave system employs denaturing HPLC to separate these homo- and hetero-duplexes by ion-pairing reverse-phase HPLC.
As an alternative, Transgenomic's Surveyor Nuclease cleaves such heteroduplexes with high specificity at sites of base mismatch or small insertions/deletions.
It is a reliable tool in analysing DNA variations, especially where the mutant alleles are at a very low concentration within the sample (less than one per cent 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 three 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 tumour analysis via surrogate tissues such as blood and urine.
