Researchers at the University of California, Irvine have developed methods to enable greater multiplexing abilities for digital polymerase chain reaction (PCR) so that up to 100 genetic targets may be analyzed. In the past multiplexing of digital PCR samples has been limited to only one probe per color. However multiple probes may be labeled by using combinatorial encoding of color, exploiting reaction rates of PCR cycles and modulating the intensity of Taqman and/or intercalating dyes therefore allowing a greater number of probes to be labeled.
Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify one or more instances of a sequence or segment of DNA across several orders of magnitude, generating millions of copies of a particular DNA sequence. It is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. Several improvements of PCR include real-time PCR (qPCR) and multiplex PCR (mPCR) that overcome difficulties associated with conventional PCR processes. qPCR allows for simultaneous amplification and quantification of DNA samples and mPCR rapidly detects multiple gene sequences using uniquely labelled primer sequences. These methods still suffer from drawbacks when faced with the high-degree multiplex PCR reactions.
Researchers at UC Irvine use digital PCR (dPCR) to overcome the previously noted problems. The process involves discretizing a larger PCR volume into several smaller reaction volumes, and limiting the number of DNA amplicons in the PCR solution to favor no more than a single amplicon per reaction volume. Therefore each reactor will have a 0 or 1 output following the PCR process. The resulting PCR amplified signal (here, fluorescence) is the product of only one amplicon and is isolated from all other discrete reactor volumes. The amplifications are quantified by counting the digital reactors with amplified fluorescence signal that corresponds to an intercalating dye or DNA polymerase probe sequence.
This invention is to be used for high density PCR technologies and real-time quantification.
100-plex digital PCR may be achieved to allow for time-saving benefits. Currently, 7-plex PCR has been demonstrated in published literature. Furthermore, both end-point and real-time analysis of droplets is possible.