METHOD OF GENERATING LONG NUCLEIC ACID MOLECULES OF DEFINED SEQUENCE
First Claim
1. A method of generating a single-stranded nucleic acid molecule, the method comprising:
- (a) combining in a mixture under conditions suitable for a polymerase extension reaction;
(i) a polymerase, (ii) an initial polynucleotide comprising a 5′
portion and a 3′
portion, wherein the polynucleotide forms the nucleic acid molecule 5′
end; and
(iii) a plurality of overlapping template oligonucleotides each having a 5′
template portion and a 3′
portion, wherein the 3′
portion of each template oligonucleotide is sufficiently complementary to the 3′
portion of one of;
the initial polynucleotide or a 3′
portion of an extension polynucleotide formed by a polymerase extension reaction of the initial polynucleotide, (b) hybridizing the initial polynucleotide or the extension polynucleotide and one of the template oligonucleotides;
(c) incubating the mixture for sufficient time to allow an extension polynucleotide to be synthesized;
(d) adding a competimer that competes with the template oligonucleotide in step (b);
(e) denaturing the extension polynucleotide and template oligonucleotide; and
(f) repeating steps (b), (c), (d), and (e) to generate the single-stranded nucleic acid molecule, wherein the number of repeated cycles equals the number of different template oligonucleotides.
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Abstract
A method of generating a single-stranded nucleic acid molecule comprising (a) combining in a mixture under conditions suitable for a polymerase extension reaction, (i) a polymerase, (ii) an initial polynucleotide comprising a 5′ portion and a 3′ portion, wherein the polynucleotide forms the nucleic acid molecule 5′ end; and (iii) a plurality of overlapping template oligonucleotides each having a 5′ template portion and a 3′ portion. The method further comprises (b) hybridizing the initial polynucleotide or the extension polynucleotide and one of the template oligonucleotides; (c) incubating the mixture for sufficient time to allow an extension polynucleotide to be synthesized; (d) adding a competimer that competes with the template oligonucleotide in step (b); (e) denaturing the extension polynucleotide and template oligonucleotide; and (f) repeating steps (b), (c), (d), and (e) to generate the single-stranded nucleic acid molecule, wherein the number of repeated cycles equals the number of different template oligonucleotides.
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Citations
52 Claims
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1. A method of generating a single-stranded nucleic acid molecule, the method comprising:
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(a) combining in a mixture under conditions suitable for a polymerase extension reaction;
(i) a polymerase, (ii) an initial polynucleotide comprising a 5′
portion and a 3′
portion, wherein the polynucleotide forms the nucleic acid molecule 5′
end; and
(iii) a plurality of overlapping template oligonucleotides each having a 5′
template portion and a 3′
portion, wherein the 3′
portion of each template oligonucleotide is sufficiently complementary to the 3′
portion of one of;
the initial polynucleotide or a 3′
portion of an extension polynucleotide formed by a polymerase extension reaction of the initial polynucleotide,(b) hybridizing the initial polynucleotide or the extension polynucleotide and one of the template oligonucleotides;
(c) incubating the mixture for sufficient time to allow an extension polynucleotide to be synthesized;
(d) adding a competimer that competes with the template oligonucleotide in step (b);
(e) denaturing the extension polynucleotide and template oligonucleotide; and
(f) repeating steps (b), (c), (d), and (e) to generate the single-stranded nucleic acid molecule, wherein the number of repeated cycles equals the number of different template oligonucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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28. A method of generating a single-stranded nucleic acid molecule, the method comprising:
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(a) combining in a mixture under conditions suitable for a polymerase extension reaction;
(i) a polymerase, (ii) an initial polynucleotide comprising a 5′
portion and a 3′
portion, wherein the polynucleotide forms the nucleic acid molecule 5′
end; and
(iii) a plurality of overlapping template oligonucleotides each having a 5′
template portion and a 3′
portion, wherein the 3′
portion of each template oligonucleotide is sufficiently complementary to the 3′
portion of one of;
the initial polynucleotide or a 3′
portion of an extension polynucleotide formed by a polymerase extension reaction of the initial polynucleotide,wherein, the concentration of each subsequent template oligonucleotide is greater than the concentration of the previous template nucleotide used to form the extension polynucleotide;
(b) hybridizing the initial polynucleotide or the extension polynucleotide and one of the template oligonucleotides;
(c) incubating the mixture for sufficient time to allow an extension polynucleotide to be synthesized;
(d) denaturing the extension polynucleotide and template oligonucleotide; and
(e) repeating steps (b), (c), and (d) to generate the single-stranded nucleic acid molecule. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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Specification