Quasi-digital PCR: Enrichment and quantification of rare DNA variants

Abstract

Rare variant enrichment and quantification was achieved by allele-specific, competitive blocker, digital PCR for aiming to provide a noninvasive method for detecting rare DNA variants from circulating cells. The allele-specific blocking chemistry improves sensitivity and lowers assay cost over previously described digital PCR methods while the instrumentation allowed for rapid thermal cycling for faster turnaround time. Because the digital counting of the amplified variants occurs in the presence of many wild-type templates in each well, the method is called "quasi-digital PCR". A spinning disk was used to separate samples into 1000 wells, followed by rapid-cycle, allele-specific amplification in the presence of a molecular beacon that serves as both a blocker and digital indicator. Monte Carlo simulations gave similar results to Poisson distribution statistics for mean number of template molecules and provided an upper and lower bound at a specified confidence level and accounted for input DNA concentration variation. A 111 bp genomic DNA fragment including the BRAF p.V600E mutation (c.T1799A) was amplified with quasi-digital PCR using cycle times of 23 s. Dilution series confirmed that wild-type amplification was suppressed and that the sensitivity for the mutant allele was<0.01 % (43 mutant alleles amongst 500,000 wild-type alleles). The Monte Carlo method presented here is publically available on the internet and can calculate target concentration given digital data or predict digital data given target concentration.

Polymerase chain reaction in microfluidic devices

“…4C). 116 The disc consisted of a central loading chamber in which the sample and oil phase were injected. It demonstrated that compartments were generated through chambers rather than droplets on a centrifugal platform.…”

Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients

“…Furthermore, a qualitative method based of the counts of positive PCRs can be applied (digital PCR). In accordance with a prior study, in which rare DNA variants were detected, this digital counting of PCR replicates with methylated ccfDNA can be called “quasi-digital PCR” [ 30 ]. On the basis of the distribution of positive and negative reactions, Poisson statistics can predict the amount of methylated ccfDNA in the sample [ 31 ].…”

Advances in Microfluidics and Lab-on-a-Chip Technologies

“…As an example, droplet-based digital PCR enabled the precise determination of mutations in several cancer cell-lines and the precise quantification of a single mutated KRAS gene in a background of 200,000 unmutated KRAS genes (Pekin, et al, 2011). In a similar vein, BRAF mutation detection was accomplished using a spinning disk digital PCR layout ( Figure 2) (Sundberg, et al, 2014). In this case a polymer microfluidic system the size of a DVD was fabricated with a single spiral channel that contained 1000 microwells on the outside of the disc.…”

“…(Inset) Post PCR fluorescent image of the disk.Fluorescence correlates to number of copies per well. Reprinted with permission from(Sundberg, et al, 2014). Copyright 2010 American Chemical Society.…”