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) 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, wherein the solid support comprises a polymeric coating polymerized from surface-modifying monomers comprising one or more monomers selected from the group consisting of dopamine, tannic acid, caffeic acid, pyrogallol, gallic acid, epigallocatechin gallate, and epicatechin gallate monomers, and wherein the primers are bound to the polymeric coating;
e) 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;
f) in a presence of at least one detergent, hybridizing a set of labeled probes to the plurality of amplification products to generate a plurality of amplification products comprising hybridized labeled probes; and
g) using imaging to count the plurality of amplification products comprising hybridized labeled probes.
2. The method of claim 1 , wherein the set of labeled probes comprises five or more different labels, wherein the counting is based on detecting the five or more different labels.
3. The method of claim 1 , wherein the at least one detergent is selected from the group consisting of anionic detergent agents; cationic detergent agents; non-ionic detergent agents; zwitterionic detergent agents; and mixtures of detergent agents.
4. The method of claim 3 , wherein the at least one detergent comprises one or more of sodium dodecyl sulfate; sodium lauryl sulfate; ammonium lauryl sulfate; benzalkonium chloride; cetyltrimethylammonium bromide; sodium dodecylbenzene sulfonate; polyoxyethylene (20) sorbitan-monolaurate; polyoxyethylene (20)-monopalmitate; polyoxyethylene (20)-monostearate; polyoxyethylene (20)-monooleate; polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether; steroid and steroidal glycosides, saponin, and digitonin; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; sodium dodecylbenzene sulfonate; and sodium C 12 -C 15 alcohol ether sulfate.
5. The method of claim 1 , wherein the plurality of primers is extended in a rolling circle amplification (RCA) reaction to form RCA product.
6. The method of claim 1 , further comprising a prior to step d) a step of treating the reaction mixture with more than one exonuclease, wherein circularized nucleic acid probes are not a substrate for the more than one exonuclease.
7. The method of claim 6 , wherein the more than one exonuclease comprise at least one exonuclease selected from the group consisting of Rec Jf, Exo VII, Exo I, and Thermolabile Exo I.
8. The method of claim 7 , wherein the more than one exonuclease comprise Rec Jf, Exo VII, and Exo I.
9. The method of claim 7 , wherein the more than one exonuclease comprise Rec Jf, Exo VII, and Thermolabile Exo I.
10. The method of claim 6 , further comprising, between c) and d), inactivating the more than one exonuclease.
11. The method of claim 1 , wherein the nucleic acid molecules comprise DNA from a sample from a subject.
12. The method of claim 11 wherein the sample from a subject is a blood or blood product sample.
13. The method of claim 1 , wherein the polymeric coating comprises tannic acid.
14. The method of claim 1 , wherein the polymeric coating is a homopolymeric coating.
15. The method of claim 1 , wherein the solid support comprises glass.