Method for the preparation of nucleic acid
First Claim
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1. A method of preparing a plurality of nucleic acid molecules, comprising:
- contacting a template nucleic acid molecule with at least a first, second and third primer and a polypeptide having DNA polymerase activity to form a mixture;
incubating the mixture under conditions sufficient to extend the primers, wherein both the second and the third primers comprise a sequence that anneals to a sequence on the template and, wherein the sequence of at least one of the second and third primers comprises at least one base that does not base pair with the sequence of the template.
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Abstract
The present invention provides materials and methods for the rapid cloning and mutagenesis of nucleic acid molecules. The present invention permits simultaneous introduction of a one or more point mutations and adapter sequences into a nucleic acid of interest.
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Citations
25 Claims
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1. A method of preparing a plurality of nucleic acid molecules, comprising:
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contacting a template nucleic acid molecule with at least a first, second and third primer and a polypeptide having DNA polymerase activity to form a mixture;
incubating the mixture under conditions sufficient to extend the primers, wherein both the second and the third primers comprise a sequence that anneals to a sequence on the template and, wherein the sequence of at least one of the second and third primers comprises at least one base that does not base pair with the sequence of the template.
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2. A method of amplifying a double-stranded DNA molecule comprising:
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(a) providing at least a first, second and third primer, wherein the first primer is at least partially complementary to a sequence of the second strand of the DNA molecule and the second and third primers are at least partially complementary to a sequence of the first strand of the DNA molecule;
(b) hybridizing the first primer to the second strand and the second and third primers to the first strand in the presence of a polypeptide having DNA polymerase activity, under conditions such that a third DNA strand complementary to the second strand and a fourth and a fifth DNA strand complementary to the first strand are synthesized, wherein at least one of the second and third primers contains nucleotide that does not base pair to the sequence on the first strand of the DNA molecule to which it hybridizes. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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Specification