METHODS FOR PREPARING CAPTURE SUBSTRATES
Methods of controlling the density or number of nucleic acid molecules coupled to solid supports are provided. Capture substrates produced thereby or comprising a predetermined density or number of nucleic acids coupled thereto are also provided.
1 - 77 . (canceled)
78 . A method of controlling density of nucleic acid molecules coupled to solid supports, the method comprising:
a. providing a solid support comprising a plurality of first coupling moieties; and
b. contacting the solid support with a plurality of nucleic acid molecules comprising second coupling moieties under conditions sufficient to couple a first coupling moiety of the plurality of first coupling moieties to a second coupling moiety to produce a coupled solid support, wherein each nucleic acid molecule comprises either:
i. a respective first strand and a respective second strand, wherein each first strand comprises a 5′ linked second coupling moiety configured to couple to the first coupling moiety, or
ii. a third strand comprising a double-stranded region in which a first region of the third strand is hybridized to a second region of the third strand, and wherein the third strand comprises a 5′ linked second coupling moiety configured to couple to the first coupling moiety;
thereby providing a coupled solid support coupled to nucleic acid molecules from the plurality of nucleic acid molecules at a density that is less than a predetermined percentage of a maximal density.
79 . The method of claim 78 , wherein the maximal density comprises all of the plurality of first coupling moieties of the solid support.
80 . The method of claim 78 , wherein the predetermined percentage is about 50%, about 60%, about 70%, about 80%, or about 90%.
81 . The method of claim 78 , wherein the density of the coupled solid support coupled to nucleic acid molecules is less than 2,500 nucleic acid molecules per square micron.
82 . The method of claim 78 , wherein, for each nucleic acid molecule, at least a portion of the respective first strand is hybridized to at least a portion of the respective second strand.
83 . The method of claim 78 , wherein the first coupling moiety does not comprise a nucleic acid.
84 . The method of claim 78 , wherein the first coupling moiety and the second coupling moiety couple by forming a covalent bond.
85 . The method of claim 84 , wherein the first coupling moiety and the second coupling moiety couple by click chemistry.
86 . The method of claim 85 , wherein the first coupling moiety is an azide molecule and the second coupling moiety is a diarylcyclooctyne moiety, or wherein the first coupling moiety is a diarylcyclooctyne moiety and the second coupling moiety is an azide molecule.
87 . The method of claim 78 , wherein the first coupling moiety comprises a nucleic acid, and the first coupling moiety and the second coupling moiety couple by forming a non-covalent bond.
88 . The method of claim 78 , wherein the third strand further comprises an unhybridized region between the first region and the second region and wherein the unhybridized region comprises at least one cleavable or excisable base.
89 . The method of claim 88 , further comprising subjecting the coupled solid support to conditions sufficient to cleave or excise the at least one cleavable or excisable base to produce two separate strands.
90 . The method of claim 88 , wherein the at least one cleavable or excisable base is selected from the group consisting of: a ribonucleic acid (RNA) base, a uracil base, an inosine base, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) base, 8-oxo-7,8-dihydroguanine (8oxoG) base, a photocleavable base, and any combination thereof.
91 . The method of claim 88 , wherein the at least one cleavable or excisable base is adjacent to a 3′ end of the first region.
92 . The method of claim 88 , wherein the unhybridized region comprises a polyT stretch of at least 5 bases.
93 . The method of claim 88 , wherein a first cleavable or excisable base is adjacent to a 3′ end of the first region and wherein a second cleavable or excisable base is adjacent to 5′ end of the second region.
94 . The method of claim 78 , wherein a nucleic acid molecule in the plurality of nucleic acid molecules coupled to the solid support comprises a primer hybridized thereto.
95 . The method of claim 94 , further comprising:
e. extending the primer from a 3′ end to produce a nucleic acid strand that is coupled to the solid support and has reverse complementary to a target nucleic acid molecule.
96 . The method of claim 95 , further comprising:
f. clonally amplifying the target nucleic acid molecule using primers coupled to the solid support.
97 . The method of claim 78 , wherein the solid support is a bead.