SURFACE-CAPTURE OF TARGET NUCLEIC ACIDS
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.
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.
2 . The method of claim 1 , wherein the linearized target nucleic acid which is captured onto the solid support is unamplified.
3 . The method of claim 1 , wherein step (a) of obtaining the target nucleic acid comprises fragmenting a starting nucleic acid to produce the target nucleic acid having at least one defined end sequence.
4 . The method of claim 3 , wherein the average length of the target nucleic acid is at least 500 nts.
5 . The method of claim 3 , wherein the target nucleic acid contains (1) a unique combination of two defined ends or (2) a unique combination of one defined end sequence and one internal sequence.
6 . The method of claim 1 , wherein step (aa) comprises digesting the starting nucleic acid with one or more restriction enzymes.
7 . The method of claim 1 , wherein step (b) of circularizing the target nucleic acid comprises:
(ba) denaturing the target nucleic acid if it is double-stranded, thereby producing a single-stranded target nucleic acid.
(bb) contacting the single-stranded target nucleic acid with a double-stranded capture probe having two overhang ends specific to two corresponding sites on the target nucleic acid;
(bc) allowing the capture probe and the target nucleic acid to anneal to each other;
(bd) optionally, cleaving any branched structures; and
(be) ligating the capture probe and the target fragment to form a partially double-stranded closed circular nucleic acid.
8 . The method of claim 7 , wherein both overhang ends of the capture probe are complementary to two respective restriction cut sites of two different restriction enzymes.
9 . The method of claim 1 , wherein step (c) of removing the linear nucleic acids comprises treating the linear nucleic acids with an exonuclease.
10 . The method of claim 1 , wherein step (d) of linearizing the target nucleic acid comprises treating the circularized target nucleic acid with a rare-cutter restriction enzyme.
11 . The method of claim 1 , wherein step (d) of linearizing the target nucleic acid comprises treating the circularized target nucleic acid with glycosylase-lyase and endonuclease.
12 . The method of claim 1 , wherein step (d) of linearizing the target nucleic acid comprises treating the circularized target nucleic acid with uracil DNA glycosylase-lyase and endonuclease VIII.
13 . The method of claim 1 , wherein step (d) of linearizing the target nucleic acid comprises randomly fragmenting the linearized or circular single-stranded nucleic acid by shearing.
14 . The method of claim 13 , wherein the random fragments produced are of sufficient length to map back to a reference sequence.
15 . The method of claim 1 , wherein step (d) of linearizing the target nucleic acid is followed by adding a capture sequence to the linearized nucleic acid(s) at the 3′ end(s) if the capture sequence is absent.
16 . The method of claim 15 , wherein the capture sequence is polyN n , wherein N is U, A, T, G, or C, and n≧5.
17 . The method of claim 1 , wherein step (d) of linearizing the target nucleic acid is followed by adding a recognition site to the linearized nucleic acid(s) at the 5′ end(s) if the recognition site is absent.
18 . The method of claim 1 , wherein in step (e) the linearized nucleic acids are bound onto the solid support by hybridizing the capture sequence to a complementary sequence covalently attached to the solid support.
19 . A method of sequencing a nucleic acid, comprising:
(i) capturing a target nucleic acid onto a solid support using the method of claim 1 ; and
(ii) sequencing the linearized nucleic acids captured on the solid support.
20 . A method of determining a nucleic acid copy number, comprising:
(i) capturing an unamplified target nucleic acid onto a solid support using the method of claim 1 ; and
(ii) determining the copy number of the linearized nucleic acids captured on the solid support.
21 . A method of capturing a nucleic acid onto a solid support, the method comprising:
(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.
22 . A nucleic acid probe comprising:
(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.
23 . The probe of claim 22 , wherein the capture sequence is polyN n , wherein N is U, A, T, G, or C, and n≧5.
24 . The probe of claim 22 , wherein the cleavage site comprises 1-10 uracils.
25 . The probe of claim 22 , wherein the probe comprises at least 1 uracil cleavage site in each strand of the double-stranded probe.