Method of detecting variation or polymorphism
First Claim
1. A method for detecting mutations and/or polymorphisms within a specific region of a target nucleotide sequence comprising the steps of:
- (1) incubating the following elements (a) to (e) under conditions that allow complementary strand synthesis wherein second and first primers are used as origins;
(a) a nucleic acid sample comprising a target nucleotide sequence;
(b) a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement;
(c) a second primer, wherein the 3′
-end of the second primer anneals to the 3′
-side region of one strand of said target nucleotide sequence and the 5′
-side of the second primer includes a nucleotide sequence complementary to the predicted nucleotide sequence that constitutes a region on the products of the complementary strand synthesis reaction that uses the second primer as the origin;
(d) a first primer, wherein the 3′
-end of the first primer anneals to the 3′
-side region of the other strand of said target nucleotide sequence, and the 5′
-side of the first primer includes a nucleotide sequence complementary to the predicted nucleotide sequence that constitutes a region on the products of the complementary strand synthesis reaction that uses the first primer as the origin; and
(e) nucleotide substrates; and
(2) correlating the amount of product synthesized by a complementary strand synthesis reaction, which uses the target nucleotide sequence as the template, with the presence of a mutation and/or polymorphism in the specific region, wherein a nucleotide sequence arranged on at least one of the 5′
-ends of the second and first primer contains a nucleotide sequence that is complementary to the predicted nucleotide sequence of said specific region or the complementary strand thereof, and wherein the complementary strand, that is synthesized using the nucleotide sequence arranged on the 5′
-end as the template and functions as the origin of complementary strand synthesis by annealing to the specific region or the complementary strand thereof, inhibits the complementary strand synthesis when the nucleotide sequence that contains the specific region is not a predicted one.
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Abstract
The method of the present invention is based on a method wherein a nucleic acid is synthesized utilizing the hybridized 3′-end, which is synthesized by complementary strand synthesis, on a specific region of a target nucleotide sequence existing as the nucleotide sequence of the same strand as the origin for the next round of complementary strand synthesis. The hybridization to a specific region, which region is searched for mutations and polymorphisms, is repeatedly carried out according to the present invention. Thus, mutations and polymorphisms in a target nucleotide sequence are exactly copied to the reaction products.
40 Citations
22 Claims
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1. A method for detecting mutations and/or polymorphisms within a specific region of a target nucleotide sequence comprising the steps of:
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(1) incubating the following elements (a) to (e) under conditions that allow complementary strand synthesis wherein second and first primers are used as origins; (a) a nucleic acid sample comprising a target nucleotide sequence; (b) a DNA polymerase catalyzing complementary strand synthesis which includes strand displacement; (c) a second primer, wherein the 3′
-end of the second primer anneals to the 3′
-side region of one strand of said target nucleotide sequence and the 5′
-side of the second primer includes a nucleotide sequence complementary to the predicted nucleotide sequence that constitutes a region on the products of the complementary strand synthesis reaction that uses the second primer as the origin;(d) a first primer, wherein the 3′
-end of the first primer anneals to the 3′
-side region of the other strand of said target nucleotide sequence, and the 5′
-side of the first primer includes a nucleotide sequence complementary to the predicted nucleotide sequence that constitutes a region on the products of the complementary strand synthesis reaction that uses the first primer as the origin; and(e) nucleotide substrates; and (2) correlating the amount of product synthesized by a complementary strand synthesis reaction, which uses the target nucleotide sequence as the template, with the presence of a mutation and/or polymorphism in the specific region, wherein a nucleotide sequence arranged on at least one of the 5′
-ends of the second and first primer contains a nucleotide sequence that is complementary to the predicted nucleotide sequence of said specific region or the complementary strand thereof, and wherein the complementary strand, that is synthesized using the nucleotide sequence arranged on the 5′
-end as the template and functions as the origin of complementary strand synthesis by annealing to the specific region or the complementary strand thereof, inhibits the complementary strand synthesis when the nucleotide sequence that contains the specific region is not a predicted one. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for detecting mutations and/or polymorphisms in a specific region of a target nucleotide sequence, wherein the nucleotide sequence arranged on at least one of the 5′
- -ends of a second and first primer contains a nucleotide sequence that is complementary to the predicted nucleotide sequence of said specific region or the complementary strand thereof, and wherein the complementary strand, that is synthesized using the nucleotide sequence arranged on the 5′
-end as the template and functions as the origin of complementary strand synthesis by annealing to the specific region or the complementary strand thereof, inhibits the complementary strand synthesis when the nucleotide sequence that contains the specific region is not a predicted one, which method comprises the steps of;a) annealing a first primer to a target nucleotide sequence to carry out a complementary strand synthesis reaction using the primer as the origin, wherein the 3′
-end of the first primer anneals to a 3′
-side region of one of said target nucleotide sequence strands, and the 5′
-side of the first primer includes a nucleotide sequence complementary to the predicted nucleotide sequence that contains a region on the product of the complementary strand synthesis reaction that uses the primer as the origin;b) putting the region, to be annealed with the second primer, in the elongation product of the first primer synthesized in step a) into a state allowing base pairing of the region to the second primer, annealing them to synthesize the complementary strand using the elongation product of the first primer as a template;
wherein the 3′
-end of the second primer anneals to the 3′
-side region of the other said target nucleotide sequence strand, and the 5′
-side of the second primer includes a nucleotide sequence complementary to the predicted nucleotide sequence that contains a region on the product of the complementary strand synthesis reaction that uses the primer as the origin;c) carrying out a complementary strand synthesis using the elongation product of the second primer itself as a template by annealing the 3′
-end of the product with said predicted nucleotide sequence to the first primer in said elongation product;d) carrying out a complementary strand synthesis using the complementary strand synthesized in step c) itself as a template by annealing the 3′
-end of the complementary strand synthesized in step c) with said predicted nucleotide sequence to the second primer in said elongation product; ande) correlating the amount of synthesized product obtained using the second primer as the origin with the presence of a mutation and/or polymorphism in the specific region. - View Dependent Claims (17, 18, 19, 20, 21, 22)
- -ends of a second and first primer contains a nucleotide sequence that is complementary to the predicted nucleotide sequence of said specific region or the complementary strand thereof, and wherein the complementary strand, that is synthesized using the nucleotide sequence arranged on the 5′
Specification