COLD-PCR (co-amplification at lower denaturation temperature-PCR is a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence (Li and Makrigiorgos 2009). For clinically relevant micro-deletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. It enriches mutations in cancer samples where normal DNA predominates. The range of enrichment demonstrated to date varies from 3 to 100-fold. Its effectiveness has been demonstrated in enriching for mutations in cancer-related genes in samples where DNA sequencing cannot detect very low concentrations of somatic DNA mutations. COLD-PCR is expected to have diverse applications in the fields of biomarker identification and tracing, genomic instability, infectious diseases, DNA methylation testing and prenatal identification of fetal alleles in maternal blood.
ddPCR for Detection of Cancer Biomarkers in Cell Free Plasma DNA
Tumor genotyping using cell free plasma DNA (cfDNA) has potential for noninvasive assessment of tumor biology, yet many existing assays are cumbersome and may give false positive results. Noninvasive genotyping of cfDNA using droplet digital PCR (ddPCR) could enable effective translation into a clinical diagnostic. Serial use of ddPCR in EGFR-mutant lung cancer patients in a prospective trial of erlotinib allowed assessment of response and resistance, including detection of resistance mutations up to 16 w prior to radiographic progression (Oxnard et al. 2014).
Comparison of miRNA quantification by ddPCR and real-time PCR has revealed greater precision and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. Applied ddPCR to serum miRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer (Hindson et al. 2013). This approach was tested in advanced prostate cancer and age-matched male controls to measure the abundance of miR- 141, which has been shown to be a biomarker for advanced prostate cancer. Compared to analysis of samples by qPCR ddPCR improved day-to-day reproducibility 7-fold. ddPCR enables accurate follow-up of serum miRNA biomarker concentrations over time during the course of treatment, which is nearly impossible to achieve with real-time PCR.