SURFACE-CAPTURE OF TARGET NUCLEIC ACIDS
First Claim
1. A method of capturing a target nucleic acid onto a solid support, the method comprising:
- (a) obtaining a sample comprising a target nucleic acid;
(b) circularizing the target nucleic acid;
(c) removing non-circularized nucleic acids;
(d) linearizing the target nucleic acid; and
(e) capturing the linearized target nucleic acid onto the solid support.
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Abstract
The disclosure provides methods of capturing target nucleic acids (e.g., gene or gene fragments) onto a solid support for further analysis. The disclosed methods utilize a capture probe that selectively circularizes only the target nucleic acid. Following the circularization of the target, the linear, non-target, nucleic acids are removed from the sample. Next, the circularized target is linearized and bound to a solid support. To allow for linearization, the capture probe may include a cleavage site that can be a noncanonical nucleotide(s) (e.g., uracil in DNA) and/or a rare-cutter site (e.g., the Not I restriction site). In some embodiments, the target nucleic acid is captured onto a support without an intermediate amplification step.
66 Citations
25 Claims
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1. A method of capturing a target nucleic acid onto a solid support, the method comprising:
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(a) obtaining a sample comprising a target nucleic acid; (b) circularizing the target nucleic acid; (c) removing non-circularized nucleic acids; (d) linearizing the target nucleic acid; and (e) capturing the linearized target nucleic acid onto the solid support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method of capturing a nucleic acid onto a solid support, the method comprising:
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(i) fragmenting a nucleic acid to produce one or more target fragments, each fragment having at least one defined end sequence; (ii) denaturing the target fragment if it is double-stranded, thereby producing a single-stranded target fragment; (iii) contacting the single-stranded target fragment with a double-stranded capture probe having two overhang ends specific to two corresponding sites on the target fragment; (iv) allowing the capture probe and the target fragment to anneal to each other; (v) optionally, cleaving any branched structures; (vi) ligating the capture probe and the target fragment to form a closed circular nucleic acid; (vii) removing remaining linear nucleic acids; (viii) optionally, denaturing the double-stranded circular nucleic acid to create a single-stranded circular nucleic acid; (ix) linearizing the single-stranded circular nucleic acid and, optionally, further fragmenting the linearized nucleic acid, or fragmenting the circular single-stranded nucleic acid; (x) adding a capture sequence at the 3′
end(s) of the linearized nucleic acid fragment(s), and optionally adding a recognition site at the 5′
end(s) of the linearized nucleic acid fragment(s); and(xi) capturing the linearized nucleic acids onto the solid support by hybridizing the capture sequence to a complementary sequence covalently attached to the solid support.
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22. A nucleic acid probe comprising:
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(a) a double-stranded nucleic acid having two overhang ends specific to two sites on a target nucleic acid, with one overhang end being complementary to a restriction cut site flanking a target sequence and the other end being complementary to a restriction cut site or an internal sequence; (b) a cleavage site within the double-stranded nucleic acid of (a), said cleavage site selected from noncanonical nucleotide(s) and a rare-cutter site; and (c) a capture sequence. - View Dependent Claims (23, 24, 25)
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Specification