Assays for single molecule detection and use thereof

Abstract

The invention relates to methods of detecting a genetic variation in a genetic sample from a subject using labeled probes and counting the number of labels in the probes.

1 . A method of detecting a genetic variation in a genetic sample from a subject comprising:
 a) contacting a first and a second probe set to the genetic sample, wherein
 (i) the first probe set comprises a first labeling probe, and a first tagging probe comprising an affinity tag, wherein the first labeling probe hybridizes adjacent to the first tagging probe on 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 tagging probe on a second 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 a portion of the first labeling probe, or complement thereof, and comprises a first label, and a second primer that hybridizes to a portion of the first tagging probe, or complement thereof, wherein the first amplified ligated probe set comprises the first label and the affinity tag, and 
 (ii) the second ligated probe set is amplified using a third primer that hybridizes to a portion of the second labeling probe, or complement thereof, and comprises a second label, and the second primer, wherein the second primer hybridizes to a portion of the second tagging probe, the second amplified ligated probe set comprises the second label and the affinity tag, and the first and second labels are different; and 
   d) immobilizing the affinity tag of the first and second amplified ligated probe sets to a pre-determined location on a substrate;   e) determining a first count of the first label immobilized on the substrate, and a second count of the second label immobilized on the substrate, wherein the first and the second labels are individually optically resolvable on the substrate; and   f) comparing the first and second counts to determine the genetic variation in the sample.   
     
     
         2 . The method of  claim 1 , wherein the first labeling probe comprises a first primer binding site, the first and second tagging probes comprise a second primer binding site, and the second labeling probe comprises a third primer binding site. 
     
     
         3 . The method of  claim 2 , wherein the first primer hybridizes to the first primer binding site, or complement thereof, the second primer hybridizes to the second primer binding site, or complement thereof, and the third primer hybridizes to the third primer binding site, or complement thereof. 
     
     
         4 . The method of  claim 1 , wherein the first nucleic acid region of interest comprises a locus on a first chromosome and the second nucleic acid region of interest comprises a locus on a second chromosome, different than the first chromosome. 
     
     
         5 . The method of  claim 4 , wherein the genetic variation is a copy number variation. 
     
     
         6 . The method of  claim 5 , wherein the genetic variation is an aneuploidy. 
     
     
         7 . The method of  claim 1 , wherein the genetic variation comprises a single nucleotide polymorphism 
     
     
         8 . The method of  claim 1 , wherein the genetic variation comprises a substitution, inversion, insertion, deletion, or mutation in a nucleotide sequence of the first or second nucleic acid region of interest. 
     
     
         9 . The method of  claim 1 , wherein the genetic sample comprises cell free DNA. 
     
     
         10 . The method of  claim 1 , wherein the subject is a pregnant female. 
     
     
         11 . The method of  claim 10 , wherein the genetic sample comprises a mixture of fetal and maternal DNA. 
     
     
         12 . The method of  claim 10 , wherein the comparing of f) determines a presence or absence of the genetic variation in a fetus. 
     
     
         13 . The method of  claim 12 , wherein the method further comprises determining a proportion of genetic material in the genetic sample derived from the fetus relative to genetic material in the sample derived from a mother of the fetus. 
     
     
         14 . The method of  claim 1 , wherein the genetic sample comprises tumor DNA and wherein the comparing of f) determines a presence or absence of a cancer in the subject. 
     
     
         15 . The method of  claim 1 , wherein the genetic sample comprises DNA from a transplanted organ and the comparing of f) determines a presence, absence or amount of DNA derived from the transplanted organ. 
     
     
         16 . The method of  claim 1 , wherein the affinity tag comprises a nucleic acid sequence. 
     
     
         17 . The method of  claim 16 , wherein the immobilizing of (d) comprises hybridizing the affinity tag to a nucleic acid sequence attached to the substrate. 
     
     
         18 . The method of  claim 1 , wherein the substrate comprises an array and the pre-determined location comprises a first member of the array. 
     
     
         19 . The method of  claim 1 , wherein the first tagging probe and the second tagging probe are the same. 
     
     
         20 . The method of  claim 1 , wherein (i) a portion of the first labeling probe does not hybridize to the first nucleic acid region of interest, (ii) a portion of the first tagging probe does not hybridize to the first nucleic acid region of interest, (iii) a portion of the second labeling probe does not hybridize to the second nucleic acid region of interest, or (iv) a portion of the second tagging probe does not hybridize to the second nucleic acid region of interest. 
     
     
         21 . The method of  claim 1 , wherein (i) the first and second nucleic acid regions of interest are at a same genetic locus, (ii) the first labeling probe hybridizes adjacent to the first tagging probe at a first allele of the genetic locus, and (iii) the second labeling probe hybridizes adjacent to the second tagging probe at a second allele of the genetic locus.