Researchers at Harvard Medical School are studying the differences in human genomes to identify the genes underlying biological processes and human disease. One of the researchers, professor Steve McCarroll, is analyzing the copy number variations (CNV) of genome segments from tens to hundreds of thousands of base pairs long. This requires the ability to measure the precise copy number of these segments in thousands of individuals.
Using technologies such as real-time PCR (qPCR) and comparative genomic hybridization arrays, the McCarroll lab could measure simple deletions and duplications—changes in copy number from two to one or zero, or from two to three or four. But what they lacked was the ability to precisely and reproducibly measure copy numbers greater than four.
Bio-Rad Laboratories’ QX100 Droplet Digital PCR (ddPCR) system provides an absolute measure of target DNA and RNA molecules. It can be used to discriminate small-fold differences in copy number, enabling researchers to measure 1, 2, 3, 4, 5, 6, or more copies.
Whereas qPCR quantifies nucleic acids by comparing the number of amplification cycles and amount of PCR end-product to those of a reference sample, ddPCR enables researchers to directly quantify nucleic acids. In other words, ddPCR does not require the use of a standard curve.
Although qPCR is a viable detection strategy when mutant and wild-type sequences are mixed, its effectiveness declines when the mutated sequence is relatively rare, such as in myleoplastic syndromes (<20–25%).
Droplet Digital PCR separates samples into 20,000 droplets, and reactions are carried out individually in each. This reduces background interference for more reliable and sensitive measurement of low concentrations of nucleic acid that may not have been detectable using qPCR.