Methods, systems, and compositions for counting nucleic acid molecules
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.
1 . A method, comprising:
a) forming at least one complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid, wherein the primer is bound to a surface on a solid support; and
b) exposing the at least one complex to a reaction mixture comprising a DNA polymerase, one or more deoxynucleoside triphosphates (dNTPs), and at least 12% polyethylene glycol (PEG), wherein the PEG has an average molecular weight between 400 and 800, under conditions wherein the primer in the complex is extended on the circularized nucleic acid by the DNA polymerase to form an extended primer.
2 . The method of claim 1 , further comprising detecting the extended primer.
3 . The method of claim 2 , wherein extension of the primer in the complex on the circularized nucleic acid is in a rolling circle amplification (RCA) reaction, wherein the extended primer comprises an RCA product.
4 . The method of claim 3 , wherein detecting the extended primer comprises:
i) hybridizing at least one probe to the RCA product; and
ii) detecting at least one hybridized probe;
wherein hybridized probe detected at a locus on the surface on the solid support is indicative of formation of a complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid at that locus on the surface of the solid support.
5 . The method of claim 4 , wherein the at least one probe comprises a labeled probe, and wherein detecting the at least one hybridized probe comprises detecting at least one hybridized labeled probe.
6 . The method of claim 5 , wherein the at least one labeled probe comprises a fluorescent label, and wherein detecting the at least one hybridized labeled probe comprises detecting fluorescence.
7 . The method of claim 6 , wherein detecting fluorescence comprises fluorescence microscopy.
8 . The method of claim 4 , wherein the at least one probe comprises a plurality of labeled probes, wherein a plurality of RCA products hybridized to the plurality of labeled probes are immobilized at loci on the surface of the solid support in a dispersal,
wherein at least a portion of the plurality of the RCA products are individually detectable at the loci on the surface of the solid support by detection of the hybridized labeled probes.
9 . The method of claim 8 , wherein the hybridized labeled probes all comprise the same label.
10 . The method of claim 8 , wherein the dispersal of RCA products is irregular.
11 . The method of claim 8 , wherein the labels on the plurality of labeled probes comprise fluorescent labels, and wherein RCA products individually detectable at loci on the surface of the solid support are detectable by detecting fluorescence.
12 . A composition comprising:
a) a plurality of complexes bound to a silanized surface of a solid support, each complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid, wherein primers in the complexes are covalently linked to the silanized surface of the solid support in a dispersal; and
b) a reaction mixture in contact with the plurality of complexes, the reaction mixture comprising a DNA polymerase, one or more dNTPs, and at least 12% PEG, wherein the PEG has an average molecular weight between 400 and 800.
13 . The composition of claim 12 , wherein the dispersal is an irregular dispersal.
14 . The composition of claim 12 , wherein the reaction mixture further comprises labeled probe.
15 . The composition of claim 14 , wherein the labeled probe comprises a fluorescent label.
16 . The composition of claim 12 , wherein the primers are covalently linked to the solid support in an irregular dispersal.
17 . The composition of claim 12 , wherein the PEG has an average molecular weight of 600.
18 . The composition of claim 12 , wherein the composition comprises at least 16% (w:v) PEG.
19 . The composition of claim 12 , wherein the composition comprises at least 18% (w:v) PEG.
20 . The composition of claim 12 , wherein the composition comprises at least 20% (w:v) PEG.