Single-stranded nucleic acid template-mediated recombination and nucleic acid fragment isolation
Methods mediated by single-stranded nucleic acid templates, including utilizing single-stranded nucleic acid templates to isolate nucleic acid fragments and to recombine nucleic acid fragments. Methods include polymerase and polymerase-free recombination of nucleic acid fragments to generate chimeric nucleic acid sequences. Integrated systems and kits are also provided.
1 - 43 . (canceled)
44 . A method of isolating nucleic acid fragments from a set of nucleic acid fragments, the method comprising:
hybridizing at least two sets of nucleic acids, wherein a first set of nucleic acids comprises single-stranded nucleic acid templates and a second set of nucleic acids comprises at least one set of nucleic acid fragments;
separating the hybridized nucleic acids from unhybridized nucleic acids by at least one first separation technique; and
denaturing the separated hybridized nucleic acids to yield the single-stranded nucleic acid templates and isolated nucleic acid fragments.
45 - 74 . (canceled)
75 . A method of generating chimeric nucleic acids, the method comprising:
hybridizing a first plurality of first parental single-stranded nucleic acids and a second plurality of second parental single-stranded nucleic acids, wherein the hybridized first and second parental single-stranded nucleic acids comprise at least one nonhybridized region of sequence diversity;
nicking at least one strand in the at least one nonhybridized region of sequence diversity;
cleaving the at least one nicked strand in the at least one nonhybridized region of sequence diversity to provide at least one sequence gap between hybridized regions; and,
elongating, ligating, or both, the at least one sequence gap between the hybridized regions to generate chimeric progeny nucleic acids.
76 . The method of claim 75 , wherein at least one of the elongating and ligating steps is conducted in vivo.
77 . The method of claim 75 , wherein at least one of the elongating and ligating steps is conducted in vitro
78 - 82 . (canceled)
83 . The method of claim 75 , comprising providing the first or second parental single-stranded nucleic acids by performing one or more cycles of an asymmetric polymerase chain reaction.
84 . The method of claim 75 , comprising providing the first or second parental single-stranded nucleic acids by degrading specific single strands in double-stranded parental sequences with at least one nuclease.
85 . (canceled)
86 . The method of claim 75 , comprising synthesizing the first or second parental single-stranded nucleic acids.
87 . The method of claim 86 , further comprising randomly or nonrandomly incorporating dUTP into the first or second parental single-stranded nucleic acids during synthesis.
88 - 93 . (canceled)
94 . The method of claim 92 , wherein the at least one nuclease comprises a Mung bean nuclease or a nickase.
95 . The method of claim 75 , the cleaving step comprising cleaving the at least one nicked strand in the at least one nonhybridized region of sequence diversity with at least one nuclease.
96 - 110 . (canceled)
111 . A method of recombining a set of nucleic acid fragments, the method comprising:
hybridizing at least two sets of nucleic acids, wherein a first set of nucleic acids comprises single-stranded sense strand-nucleic acid templates and a second set of nucleic acids consists essentially of single-stranded antisense strand-nucleic acid fragments; and,
elongating, ligating, or both, sequence gaps between the hybridized nucleic acid fragments to generate at least substantially full-length chimeric nucleic acid sequences that correspond to the single-stranded nucleic acid templates, thereby recombining the set of nucleic acid fragments.
112 - 115 . (canceled)
116 . A method of recombining a set of nucleic acid fragments, the method comprising:
providing a set of at least partially double-stranded nucleic acids that encode a polypeptide of interest or portion thereof;
contacting the set of at least partially double-stranded nucleic acids with an exonuclease that selectively degrades one strand of the at least partially double-stranded nucleic acids to provide a set of single-stranded nucleic acid templates;
hybridizing the set of single-stranded nucleic acid templates with a second set of nucleic acids comprising at least one set of nucleic acid fragments; and,
elongating, ligating, or both, sequence gaps between the hybridized nucleic acid fragments to generate at least substantially full-length chimeric nucleic acid sequences that correspond to the single-stranded nucleic acid templates, thereby recombining the set of nucleic acid fragments.
117 . The method of claim 116 , wherein the exonuclease is selected from the group consisting of Exonuclease II, Bal31, Mung bean nuclease, T7 gene 6 exonuclease, and lambda exonuclease.
118 . The method of claim 116 , wherein the nucleic acid fragments are single stranded.
119 - 166 . (canceled)