Methods, systems, and compositions for counting nucleic acid molecules

Abstract

Compositions and methods, systems, and kits for detecting and quantifying variations in numbers of molecules, particularly variations in gene dosage, e.g., due to gene duplication, or to variations from the normal euploid complement of chromosomes, e.g., trisomy of one or more chromosomes that are normally found in diploid pairs, without digital sequencing.

We claim: 
     
         1 . A method for nucleic acid molecule analysis, comprising:
 a) contacting a sample comprising nucleic acid molecules with a plurality of molecular inversion probes (MIPs), wherein the plurality of MIPs comprises a first MIP that specifically binds chromosome 13, a second MIP that specifically binds chromosome 18, a third MIP that specifically binds chromosome 21, a fourth MIP that specifically binds chromosome X, and a fifth MIP that specifically binds chromosome Y;   b) using ligation to circularize MIPs of the plurality of MIPS that hybridize to the nucleic acid molecules, thereby forming a plurality of circularized nucleic acid probes;   c) providing a solid support comprising a plurality of primers bound to a surface of the solid support;   d) hybridizing the circularized nucleic acid probes to the plurality of primers immobilized to the surface of the solid support to form immobilized complexes;   e) extending the plurality of primers in the immobilized complexes in a rolling circle amplification (RCA) reaction to form a plurality of RCA products immobilized to the solid support;   f) hybridizing a plurality of labeled oligonucleotide probes to the plurality of labeled RCA products immobilized on the solid support to generate a plurality of RCA products comprising hybridized labeled oligonucleotide probes immobilized on the solid support, wherein the plurality of labeled oligonucleotide probes comprises more than two different labels;   g) detecting the hybridized labeled oligonucleotide probes immobilized on the solid support, wherein at least a portion of the plurality of the RCA products comprising hybridized labeled oligonucleotide probes immobilized on the solid support are individually detectable by detection of the hybridized labeled oligonucleotide probes; and   h) counting the plurality of RCA products hybridized to the plurality of hybridized labeled oligonucleotide probes immobilized on the solid support based on the detecting.   
     
     
         2 . The method of  claim 1 , wherein the plurality of RCA products immobilized to the solid support is in a solution comprising a crowding agent. 
     
     
         3 . The method of  claim 2 , wherein the RCA reaction forming the plurality of RCA products immobilized to the solid support is in a solution comprising a crowding agent. 
     
     
         4 . The method of  claim 1 , wherein the surface comprises an acrylic group. 
     
     
         5 . The method of  claim 1 , wherein the solid support comprises glass. 
     
     
         6 . The method of  claim 1 , wherein the solid support comprises an assay plate. 
     
     
         7 . The method of  claim 6 , wherein the assay plate is a multi-well assay plate. 
     
     
         8 . The method of  claim 1 , wherein the plurality of primers are bound directly to the surface of the solid support. 
     
     
         9 . The method of  claim 1 , wherein the crowding agent comprises polyethylene glycol (PEG). 
     
     
         10 . The method of  claim 9 , wherein the PEG has an average molecular weight between 200 and 8000. 
     
     
         11 . The method of  claim 9 , wherein the PEG is at a concentration of at least 12%. 
     
     
         12 . The method of  claim 1 , wherein b) is performed in a reaction mixture comprising Phi29 DNA polymerase. 
     
     
         13 . The method of  claim 1 , wherein the plurality of labeled oligonucleotide probes comprise fluorescent labels. 
     
     
         14 . The method of  claim 13 , wherein the detecting comprises detecting fluorescence. 
     
     
         15 . The method of  claim 14 , wherein detecting fluorescence comprises fluorescence microscopy. 
     
     
         16 . The method of  claim 1 , wherein the plurality of primers is in a dispersal on the solid support. 
     
     
         17 . The method of  claim 1 , wherein the more than two different labels comprise more than two different fluorescent dyes.