Genomic DNA is an ideal tool to establish the limit of detection of molecular assays, but to be effective it must be extracted from cell lines with precisely controlled copy numbers and allelic frequencies, which is not easily achievable with patient derived cell lines. HDx Reference Standards therefore represent an ideal solution, due to the highly characterized, reproducible nature of the cell lines used to generate the DNA.
DNA Reference Standards are derived from engineered mutant (50% mutant) and matched “normal” (0% mutant) cell lines. Mutant cell lines are all heterozygotes, and in the majority of cases this means there is a single mutant allele and a single normal allele.
Where the copy number is 2N, a 100% mutant DNA sample indicates that 100% of the cells that the DNA was extracted from contained the mutation, however as only one of the two copies of the gene from that cell will actually contain the mutation, the sample will actually have an allelic frequency of 50% for that mutation. Genotypes with a copy number of 3N and with a single mutant allele would have an allelic frequency of 33%. Mutant and “normal” DNA can be precisely blended to generate samples with specific allelic frequencies.
|Figure 1. Using genomic DNA in the workflow to control variability|