Studying copy number variations using a nanofluidic platform

Qin, Jian; Jones, Robert C.; Ramakrishnan, Ramesh

In this study we demonstrate the use of a unique integrated nanofluidic system, the digital array, in the study of CNVs. The digital array (14, 15) is able to accurately quantitate DNA samples based on the fact that single DNA molecules are randomly distributed in more than 9000 reaction chambers and then PCR amplified. The concentration of any sequence in a DNA sample (copies/ml) can be calculated using the numbers of positive chambers that contain at least one copy of that sequence. In order to ensure that the apparent difference in gene copy numbers in different samples are real, and not distorted by differences in sample amounts, we use the expression 'relative copy number'. The relative copy number of a gene is the number of copies of that gene per haploid genome. It can be easily expressed as the ratio of the copy number of a target gene to the copy number of a single copy reference gene (two copies per cell) in a DNA sample, which is always 1 per haploid genome. By using two assays for the two genes (the gene of interest and the reference gene) with two fluorescent dyes on the same digital array, we are able to simultaneously quantitate both genes in the same DNA sample. The ratio of the numbers of molecules of these two genes is the relative copy number of the gene of interest in a DNA sample. A single copy gene should have a relative copy number of 1. A relative copy number greater than 1 indicates the presence of duplication of the target gene while a number smaller than 1 implies deletion of this gene.