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 for nucleic acid molecule analysis, comprising:
a) providing a plurality of molecular inversion probes (MIPs) and a sample of nucleic acid molecules;
b) hybridizing MIPs of the plurality of MIPs to the nucleic acid molecules to generate hybridized MIPs;
c) in a reaction mixture, circularizing hybridized MIPs to form a plurality of circularized nucleic acid probes;
d) treating the reaction mixture with more than one exonuclease, wherein circularized nucleic acid probes are not a substrate for the more than one exonucleases;
e) forming a plurality of complexes comprising a plurality of primers hybridized to a plurality of circularized nucleic acid probes from the reaction mixture following d), wherein the plurality of primers is bound to a solid support;
f) extending the plurality of primers in the plurality of complexes in an amplification reaction to form a plurality of amplification products immobilized to the solid support;
g) in a presence of a detergent, hybridizing a set of labeled probes to the plurality of amplification products to generate a plurality of amplification products comprising hybridized labeled probes, wherein the set of labeled probes comprises five or more different labels; and
h) using imaging to count the plurality of amplification products comprising hybridized labeled probes based on detecting the five or more different labels.
2. The method of claim 1 , wherein the more than one exonucleases comprise Rec Jf.
3. The method of claim 1 , wherein the more than one exonucleases comprise Exo VII.
4. The method of claim 1 , wherein the more than one exonucleases comprise Exo I.
5. The method of claim 4 , wherein the more than one exonucleases comprise Thermolabile Exo I.
6. The method of claim 1 , wherein the more than one exonucleases comprise Rec Jf, Exo VII, and Exo I.
7. The method of claim 1 , wherein the more than one exonucleases comprise Rec Jf, Exo VII, and Thermolabile Exo I.
8. The method of claim 1 , further comprising, between d) and e), inactivating the more than one exonucleases.
9. The method of claim 8 , wherein the inactivating comprises heat-inactivating.
10. The method of claim 1 , wherein the solid support comprises tannic acid.
11. The method of claim 1 , wherein the solid support comprises acrylic acid.
12. The method of claim 1 , wherein the solid support comprises a homopolymeric coating.
13. The method of claim 1 , wherein the plurality of primers is bound to the solid support by a 5′ terminal amine modification.
14. The method of claim 1 , wherein the plurality of primers is covalently bound to the solid support.
15. The method of claim 1 , wherein the solid support comprises glass.
16. The method of claim 1 , wherein the solid support comprises an assay plate.
17. The method of claim 16 , wherein the assay plate is a multi-well assay plate.
18. The method of claim 1 , wherein in f) the plurality of amplification products immobilized to the solid support are in a solution comprising a crowding agent.
19. The method of claim 18 , wherein the crowding agent comprises polyethylene glycol (PEG).
20. The method of claim 19 , wherein the PEG has an average molecular weight between 200 and 8000.
21. The method of claim 19 , wherein the PEG is at a concentration of at least 12%.
22. The method of claim 1 , wherein the plurality of primers are bound to the solid support in an irregular dispersal.
23. The method of claim 1 , wherein the five or more different labels comprise five or more different fluorescent labels.