Patent ID: 8722334

Claim:
A method for determining in a sample taken from a woman pregnant with a fetus the relative amount of a target gene derived from the woman and from the fetus, comprising the steps of: (i) preparing multiple equal fractions from the sample, wherein more than 50% of the fractions contain no more than 1 target gene molecule per fraction; (ii) treating each fraction with a methylation-sensitive restriction enzyme, which cleaves the target gene at a predetermined location when there is no methylation at the location and does not cleave the target gene at the location when there is methylation at the location; (iii) performing amplification reactions in each fraction using at least one forward primer with at least two reverse primers, or using at least two forward primers with at least one reverse primer, wherein the predetermined location is between at least one pair of the forward and reverse primers but is not between at least one, different, pair of forward and reverse primers; (iv) detecting in each fraction amplified nucleotide sequence from each pair of forward and reverse primers; and (v) counting the number of fractions in which different combinations of amplified nucleotide sequences from different pairs of forward and reverse primers are detected, thereby determining the relative amount of the target gene derived from the woman and the fetus in the sample.