| 主权项 |
1. A method of identifying target nucleic acid sequences in a sample, comprising:
(a) generating from a sample a plurality of first template molecules and a plurality of second template molecules, wherein said first template molecules comprise a sequence of a first target nucleic acid and a first identity sequence (IS) tag, and said second template molecules comprise a sequence of a second target nucleic acid and a second IS tag, and wherein said first IS tag comprises a first identification (ID) code and said second IS tag comprises a second ID code; (b) generating at least one cluster of nucleic acid amplicons by nucleic acid amplification of said first template molecules and at least one cluster of nucleic acid amplicons by clonal amplification of said second template molecules in parallel individual reactions on a surface, wherein said clusters of nucleic acid amplicons are spatially separated; and (c) identifying said ID codes of said IS tags of said nucleic acid amplicons on said surface to determine the target nucleic acid sequences represented by said nucleic acid amplicons and the step (c) of identifying said ID codes of said IS tags of said nucleic acid amplicons further comprises:
(c-1) hybridizing a pair of labeled IS probes (LISPs) from a pool of LISPs with said clusters of nucleic acid amplicons, thereby said pair of LISPs are juxtaposed, wherein each said LISP comprises: (i) a sequence complimentary to the IS tag sequence it hybridizes to, and (ii) a detectable label associated with a designated base at base interrogation positions, and wherein there are two sets of the LISPs in each pool of LISPs, with one set being the 3′ labeled IS probes (3′-LISPs) that comprise 5′ phosphate groups and 3′ detectable labels, and the other set being the 5′ labeled IS probes (5′-LISPs) that comprise 5′ detectable labels and 3′ hydroxyl groups;(c-2) performing a paired-probe ligation of said pair of juxtaposed LISPs;(c-3) detecting the presence of said detectable labels on said ligated pair of LISPs on said IS tags of said nucleic acid amplicons, and elucidating the base composition at said base interrogation positions of said IS tags according to the detectable label combination of said ligated pair of LISPs, wherein two specified base positions on said IS tags and one on each side of said paired probes are interrogated;(c-4) denaturing said ligated pair of LISPs from said analyte molecule;(c-5) repeating steps (c-1) to (c-4) until all bases of base interrogation positions in said IS tags are interrogated without duplication and all base composition of said IS tags are elucidated; and(c-6) determining the ID codes of said IS tags on said nucleic acid amplicons by comparing said base composition to designated ID codes, wherein 10^n (or 10n) kinds of the base combinations are distinguished in n times of the paired-probe ligation. |
