Assays for single molecule detection and use thereof

A method of detecting a genetic variation in a fetus comprising:

• a) contacting a first and a second probe set to a sample comprising a mixture of genetic material derived from the fetus and derived from a mother of the fetus, wherein
• (i) the first probe set comprises a first labeling probe, and a first tagging probe comprising an affinity tag, wherein the affinity tag comprises a nucleic acid sequence, and the first labeling probe hybridizes adjacent to the first labeling probe at a first allele of a first nucleic acid region of interest, and
• (ii) the second probe set comprises a second labeling probe, and a second tagging probe comprising the affinity tag, wherein the second labeling probe hybridizes adjacent to the second labeling probe at a second allele of the first nucleic acid region of interest;
• b) ligating the first labeling probe to the first tagging probe, thereby providing a first ligated probe set, and ligating the second labeling probe to the second tagging probe, thereby providing a second ligated probe set;
• c) amplifying the first and second ligated probe sets to form first and second amplified ligated probe sets, respectively, wherein,
• (i) the first ligated probe set is amplified using a first primer that hybridizes to the first labeling probe, or to a complement of the first labeling probe, and comprises a first label, and a second primer that hybridizes to the first tagging probe, or to a complement of the first tagging probe, wherein the first amplified probe set comprises the first label and the affinity tag, or a complement of the affinity tag, and
• (ii) the second ligated probe set is amplified using a third primer that hybridizes to the second labeling probe, or to a complement of the second labeling probe, and comprises a second label, and the second primer, wherein the second primer hybridizes to the second tagging probe, or to a complement of the second tagging probe, wherein the second amplified probe set comprises the second label and the affinity tag, or a complement the affinity tag, and the first and second labels are different;
• and
• d) immobilizing the affinity tag, or a complement of the affinity tag, of the first and second amplified ligated probe sets to a first member of an array having a pre-defined location on the array;
• e) counting a number of individual labeled molecules of the first amplified ligated probe set immobilized on the first member thereby providing a first count, and counting a number of individual labeled molecules of the second amplified ligated probe set immobilized on the first member thereby providing a second count, wherein each of the first and the second labels are individually optically resolvable on the first member;
• and
• f) determining a presence or absence of the genetic variation in the fetus according to at least the first count and the second count.