CELL FREE CLONING OF NUCLEIC ACIDS
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Abstract
Methods and devices for cell-free sorting and cloning of nucleic acid libraries are provided herein.
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Citations
121 Claims
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1-93. -93. (canceled)
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94. A method for nucleic acid sorting comprising:
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(a) providing a plurality of circular double-stranded nucleic acids, each of the plurality of circular double-stranded nucleic acids comprising a first strand that is a continuous circle and a second strand that comprises a gap, wherein the gap has a length of at least one base; (b) diluting the plurality of circular double-stranded nucleic acids to a concentration of less than 100 nM; (c) extending the second strand a first amplification reaction, wherein the first strand is a template strand, thereby forming a plurality of amplicon nucleic acids comprising a plurality of copies of the first strand; and (d) partitioning such that on average there are 0.1 to 10 amplicon nucleic acids per fraction. - View Dependent Claims (95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110)
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111. A method for nucleic acid sorting comprising:
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(a) providing a plurality of circular double-stranded nucleic acids, each of the plurality of circular double-stranded nucleic acids comprising a first strand that is a continuous circle and a second strand comprising a gap, wherein the gap has a length of at least one base; (b) partitioning such that on average there are about 0.1 to 10 circular double-stranded nucleic acids from the plurality of circular double-stranded nucleic acids per fraction; and (c) amplifying the partitioned circular double-stranded nucleic acids in the presence of a random primer to generate a plurality of amplicon nucleic acids, wherein the random primer comprises 4 to 8 bases in length. - View Dependent Claims (112, 113, 114, 115, 116, 117, 118, 119, 120)
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121. A method for nucleic acid sorting comprising:
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(a) forming a plurality of circular single-stranded nucleic acids by joining a double-stranded non-circularized nucleic acid and two adaptor sequences, wherein each of the two adaptor sequences encodes for a hairpin secondary structure; (b) diluting the plurality of circular single-stranded nucleic acids to a concentration of at most 1 nM; (c) amplifying the plurality of circular single-stranded nucleic acids in the presence of a primer having sequence complementary to one of the two adaptor sequences; and (d) partitioning the amplification reaction such that on average there are 0.1 to 10 amplicon nucleic acids per fraction.
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Specification