	US 11,739,371 B2
	Arrays for single molecule detection and use thereof
Patrick James Collins, San Francisco, CA (US); Adrian Nielsen Fehr, San Francisco, CA (US); Jill Lyndon Herschleb, San Francisco, CA (US); and Hywel Bowden Jones, San Francisco, CA (US)
Assigned to Invitae Corporation, San Francisco, CA (US)
Appl. No. 15/551,788
Filed by Invitae Corporation, San Francisco, CA (US)
PCT Filed Feb. 18, 2016, PCT No. PCT/US2016/018549 § 371(c)(1), (2) Date Aug. 17, 2017, PCT Pub. No. WO2016/134191, PCT Pub. Date Aug. 25, 2016.
Claims priority of provisional application 62/117,942, filed on Feb. 18, 2015.
Prior Publication US 2018/0023124 A1, Jan. 25, 2018
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6827 (2018.01); C07H 21/04 (2006.01)

CPC C12Q 1/6827 (2013.01) [C12Q 2565/50 (2013.01)]

10 Claims

1. A method of producing a molecular array, the method comprising:

(i) hybridizing first and second probe sets to first and second nucleic acid regions of interest in target nucleic acid sequences, respectively, in a genetic sample from a human, wherein nucleotide sequences of the first and second nucleic acid regions of interest are different,

wherein the first probe set comprises a first labeling probe comprising a first priming sequence, and a first tagging probe comprising an affinity tag and a second priming sequence, wherein the first labeling probe hybridizes adjacent to the first tagging probe on the first nucleic acid region of interest, wherein the affinity tag comprises a nucleotide sequence complementary to a capture probe immobilized to a location on a substrate, and wherein the nucleotide sequence of the affinity tag does not hybridize to the first or second nucleic acid regions of interest, and

the second probe set comprises a second labeling probe comprising a third priming sequence, and a second tagging probe comprising the affinity tag and the second priming sequence, wherein the second labeling probe hybridizes adjacent to the second tagging probe on the second nucleic acid region of interest;

(ii) 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;

(iii) amplifying the first and the second ligated probe sets to form first and second amplified ligated probe sets, respectively, wherein

(a) the first ligated probe set is amplified using:

a first primer that comprises a first label and that hybridizes to the first priming sequence or complement thereof, and

a second primer that hybridizes to the second priming sequence or complement thereof,

wherein the first amplified ligated probe set comprises a first plurality of identical single-stranded amplicons of the first ligated probe set, each comprising the first label and the affinity tag, and

(b) the second ligated probe set is amplified using:

a third primer that comprises a second label and that hybridizes to the third priming sequence or complement thereof, and

the second primer of (a),

wherein the second amplified ligated probe set comprises a second plurality of identical single-stranded amplicons of the second ligated probe set, each comprising the second label and the affinity tag, and wherein the first and second labels are different; and

(iv) immobilizing the first and second plurality of identical single-stranded amplicons to the location on the substrate by hybridizing the nucleotide sequence of the affinity tag to the capture probe immobilized on the substrate at a density wherein individual labeled molecules of the single-stranded amplicons optically resolvable at the location by digital imaging, thereby producing the molecular array comprising the first and second plurality of identical single-stranded amplicons immobilized to the location on the substrate.