Methods for high fidelity production of long nucleic acid molecules
First Claim
1. A method for generating a long nucleic acid molecule, the method comprising the steps of:
- a) providing a plurality of nucleic acids immobilized on a surface, wherein said plurality of nucleic acids comprises nucleic acids having overlapping complementary sequences, wherein said overlapping complementary sequences are capable of hybridizing to each other;
b) releasing said plurality of nucleic acids into a solution; and
c) producing the long nucleic acid molecule by the steps of;
i) forming a plurality of hybridized nucleic acids by hybridizing said overlapping complementary sequences to each other;
ii) synthesizing a plurality of intermediate-length nucleic acid duplexes by extending said plurality of hybridized nucleic acids;
iii) forming intermediate-length nucleic acids having overlapping complementary sequences by disassociating said plurality of intermediate-length nucleic acid duplexes, wherein the overlapping complementary sequences in said intermediate-length nucleic acids are capable of hybridizing to each other;
iv) forming a plurality of intermediate-length hybridized nucleic acids by hybridizing the intermediate-length nucleic acids having overlapping complementary sequences to each other;
v) synthesizing a plurality of extended intermediate-length nucleic acid duplexes by extending said plurality of intermediate-length hybridized nucleic acids;
vi) generating a plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule by repeating steps iii) to v) for multiple cycles until said plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule are produced, wherein, in each of the cycles, the plurality of intermediate-length nucleic acid duplexes in step iii) is replaced with the plurality of extended intermediate-length nucleic acid duplexes produced in step v); and
vii) forming the long nucleic acid molecule by ligating the plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule.
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Abstract
In a method for generating a long nucleic acid molecule, nucleic acids immobilized on a surface and having overlapping complementary sequences is released into solution. The overlapping complementary sequences are hybridized to form hybridized nucleic acids, followed by extension or ligation of the hybridized nucleic acids to synthesize the long nucleic acid molecule. The nucleic acids may comprise first and second series of nucleic acids having redundant overlapping sequences, wherein nucleic acids from the first and second series are complementary to each other. The complementary nucleic acids are hybridized to form the hybridized nucleic acids. The generated long nucleic acid molecule may have a predetermined sequence element, and it may be introduced into a system wherein the predetermined sequence element is required for replication, such that replication of the synthesized long nucleic acid molecule is indicative of the presence of the predetermined sequence element in the long nucleic acid molecule.
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Citations
14 Claims
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1. A method for generating a long nucleic acid molecule, the method comprising the steps of:
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a) providing a plurality of nucleic acids immobilized on a surface, wherein said plurality of nucleic acids comprises nucleic acids having overlapping complementary sequences, wherein said overlapping complementary sequences are capable of hybridizing to each other; b) releasing said plurality of nucleic acids into a solution; and c) producing the long nucleic acid molecule by the steps of; i) forming a plurality of hybridized nucleic acids by hybridizing said overlapping complementary sequences to each other; ii) synthesizing a plurality of intermediate-length nucleic acid duplexes by extending said plurality of hybridized nucleic acids; iii) forming intermediate-length nucleic acids having overlapping complementary sequences by disassociating said plurality of intermediate-length nucleic acid duplexes, wherein the overlapping complementary sequences in said intermediate-length nucleic acids are capable of hybridizing to each other; iv) forming a plurality of intermediate-length hybridized nucleic acids by hybridizing the intermediate-length nucleic acids having overlapping complementary sequences to each other; v) synthesizing a plurality of extended intermediate-length nucleic acid duplexes by extending said plurality of intermediate-length hybridized nucleic acids; vi) generating a plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule by repeating steps iii) to v) for multiple cycles until said plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule are produced, wherein, in each of the cycles, the plurality of intermediate-length nucleic acid duplexes in step iii) is replaced with the plurality of extended intermediate-length nucleic acid duplexes produced in step v); and vii) forming the long nucleic acid molecule by ligating the plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification