Digital analysis of nucleic acid modification

A method of obtaining a normalized ratio between a fetal nucleic acid and a maternal reference nucleic acid in a mixture that provides a percent of the fetal nucleic acid in the mixture, the method comprising:

• preparing a mixture comprising (i) a fetal nucleic acid comprising only recognition sequences that are unmethylated and is capable of cleaving by a restriction enzyme that cleaves only the recognition sequences that are unmethylated, (ii) a maternal reference nucleic acid comprising only the recognition sequences that are methylated and is not capable of cleaving by the restriction enzyme that cleaves only the recognition sequences that are unmethylated, and (iii) a known initial amount of control nucleic acid having only the recognition sequences that are unmethylated and is capable of cleaving by the restriction enzyme that cleaves only the recognition sequences that are unmethylated;
• conducting a methylation-specific enzymatic reaction by exposing the mixture to the restriction enzyme that cleaves only the recognition sequences that are unmethylated;
• partitioning the mixture into droplets after the conducting step;
• measuring an amount of the fetal nucleic acid, an amount of the maternal reference nucleic acid, and an amount of the control nucleic acid in each of the droplets that remain intact by performing a digital polymerase chain reactions (PCR) in each of the droplets, wherein the measuring step includes the use of detectably labeled probes specifically hybridized to the fetal nucleic acid, the maternal reference nucleic acid, and the control nucleic acid;
• determining an efficiency value for the methylation-specific enzymatic reaction based on the known initial amount of the control nucleic acid and the amount of the control nucleic acid determined in the measuring step;
• and
• normalizing, using the efficiency value, the amount of the fetal nucleic acid and the amount of the maternal reference nucleic acid in the mixture, thereby obtaining the normalized ratio between the fetal nucleic acid and the maternal reference nucleic acid in the mixture that provides the percent of the fetal nucleic acid in the mixture.