METHOD FOR DETECTING MUTATIONS AND/OR POLYMORPHISMS

• US 20070238113A1
• Filed: 12/22/2006
• Published: 10/11/2007
• Est. Priority Date: 11/08/1999
• Status: Active Grant

• Current Assignee: Eiken Kagaku Kabushiki Kaisha
• Original Assignee: Eiken Kagaku Kabushiki Kaisha
• Inventors: Notomi, Tsugunori, Kanda, Hidetoshi, Nagamine, Kentaro, Yonekawa, Toshihiro
• Granted Patent: US 7,638,280 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

1. A kit for detecting a mutation and/or polymorphism of a specific region in a target nucleotide sequence comprising the following elements (i)-(iv), wherein a nucleotide sequence arranged on at least one of the 5′

-ends of a second and first primer contains a nucleotide sequence complementary to the predicted nucleotide sequence of said specific region or the complementary strand thereof, further wherein the complementary strand, that is synthesized using the nucleotide sequence arranged on this 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 and when the nucleotide sequence that contains the specific region is not a predicted one;

i) a second primer, wherein the 3′

-end of the second primer anneals to the 3′

-side region of one of said target nucleotide sequence strands, and the 5′

-side of the second primer includes a nucleotide sequence complementary to the predicted nucleotide sequence that constitutes a region on the product of the complementary strand synthesis reaction that uses this primer as the origin;

ii) a first primer, wherein the 3′

-end of the first primer anneals to the 3′

-side region of the other said target nucleotide sequence strand, 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;

iii) a DNA polymerase catalyzing complementary strand synthesis which includes a strand displacement; and

iv) nucleotide substrates.

2. The kit of claim 1 additionally comprising the following elements:

v) a third primer that serves as the origin of the complementary strand synthesis by annealing to the 3′

-side of the first primer annealing region in the template; and

vi) a fourth primer that serves as the origin of the complementary strand synthesis by annealing to the 3′

-side of the second primer annealing region in the template.

3. The kit of claim 1 additionally comprising the elements of:

vii) a fifth primer amplifying an internal standard, wherein the 3′

-end of the fifth primer anneals to the 3′

-side region of one of the target nucleotide sequence strands of an internal standard, and the 5′

-side of the fifth primer includes a nucleotide sequence complementary to a nucleotide sequence that contains an arbitrary region of a product of the complementary strand synthesis reaction that uses the primer as the origin; and

iix) a sixth primer amplifying an internal standard, wherein the 3′

-end of the sixth primer anneals to the 3′

-side region of one of the target nucleotide sequence strands of an internal standard, and the 5′

-side of the sixth primer includes a nucleotide sequence complementary to the nucleotide sequence that contains a region of a product of the complementary strand synthesis reaction that uses the primer as the origin.

4. The kit of claim 3 additionally comprising the elements of:

ix) a seventh primer that serves as the origin of the complementary strand synthesis by annealing to the 3′

-side of the sixth primer annealing region in the template; and

x) a eighth primer that serves as the origin of the complementary strand synthesis by annealing to the 3′

-side of the sixth primer annealing region in the template.