Analysis of nucleic acids

A method of detecting variations in copy number of a target nucleic acid comprising:

• a. obtaining a sample comprising (i) a plurality of polynucleotides, wherein at least one of the polynucleotides comprises multiple copies of a target nucleic acid;
• (ii) a probe with a fluorescent label to detect the target nucleic acid;
• and (iii) reagents to enable a PCR reaction;
• b. separating the sample into a plurality of spatially-isolated partitions, wherein the spatially-isolated partitions comprise on average less than five copies of the target nucleic acid;
• c. subjecting the sample to a PCR amplification reaction in order to detect the target nucleic acid;
• d. detecting the fluorescence intensity of the fluorescent label before any of the reagents for the PCR reaction become limiting;
• e. enumerating the number of partitions that have a fluorescence intensity above a threshold value indicative of one copy of the target nucleic acid;
• f. enumerating the number of partitions that have a fluorescence intensity above a threshold value indicative of multiple copies of the target nucleic acid;
• and
• g. calculating the copy number of the target nucleic acid based on the numbers obtained in step e and step f.