Method for detecting a target nucleic acid sequence

• US 20070218464A1
• Filed: 03/20/2006
• Published: 09/20/2007
• Est. Priority Date: 03/20/2006
• Status: Active Grant

• Current Assignee: Toshiba Medical Systems Corporation (Canon Inc.)
• Original Assignee: Kabushiki Kaisha Toshiba (Toshiba Corporation)
• Inventors: Hashimoto, Koji, Nakamura, Naoko
• Granted Patent: US 7,488,581 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6813 Hybridisation assays

Abstract

A method of detecting a target nucleic acid sequence comprising providing a stem-and-loop structured nucleic acid for measurement wherein the nucleic acid comprises complementary sequence portions located at both terminals and a target sequence portion therebetween as well as a double-stranded portion formed by hybridization of the complementary sequence portions located at both terminals and a remaining looped single-stranded portion, providing a probe nucleic acid having a sequence complementary to the target sequence portion wherein one end of the probe nucleic acid being immobilized to a solid substrate surface, reacting the nucleic acid for measurement with the probe nucleic acid to specifically hybridize the target sequence portion of the nucleic acid for measurement to the probe nucleic acid, and detecting presence or absence of the nucleic acid for measurement hybridized to the probe nucleic acid.

Claims

1. A method of detecting a target nucleic acid sequence comprising:

providing a stem-and-loop structured nucleic acid for measurement wherein the nucleic acid comprises complementary sequence portions located at both terminals and a target sequence portion therebetween as well as a double-stranded portion formed by hybridization of the complementary sequence portions located at both terminals and a remaining looped single-stranded portion;

providing a probe nucleic acid having a sequence complementary to the target sequence portion wherein one end of the probe nucleic acid being immobilized to a solid substrate surface;

reacting the nucleic acid for measurement with the probe nucleic acid to specifically hybridize the target sequence portion of the nucleic acid for measurement to the probe nucleic acid; and

detecting presence or absence of the nucleic acid for measurement hybridized to the probe nucleic acid.

2. A method according to claim 1, wherein 5′

to 3′

sequence orientations of both the probe nucleic acid and the target sequence portion are arranged so that the double-stranded portion of the nucleic acid for measurement extends away from the solid surface when the probe nucleic acid and the target sequence portion is hybridized.

3. A method according to claim 1, wherein the nucleic acid for measurement and the probe nucleic acid are DNA.

4. A method according to claim 2, wherein the nucleic acid for measurement and the probe nucleic acid are DNA.

5. A method according to claim 1, wherein the probe nucleic acid is a component of a DNA chip.

6. A method according to claim 2, wherein the probe nucleic acid is a component of a DNA chip.

7. A method according to claim 1, wherein the nucleic acid for measurement is a LAMP product amplified by LAMP method.

8. A method according to claim 2, wherein the nucleic acid for measurement is a LAMP product amplified by LAMP method.

9. A method according to claim 7, wherein the target sequence in the LAMP product is inserted between F1 region and F2 region, between F2c region and F1c region, between B1 region and B2 region, and/or between B2c region and B1c region of the LAMP product.

10. A method according to claim 8, wherein the target sequence in the LAMP product is inserted between F1 region and F2 region, between F2c region and F1c region, between B1 region and B2 region, and/or between B2c region and B1c region of the LAMP product.

11. A method according to claim 9, wherein a part of the target sequence overlaps with a part of the sequence of the F2 region, F2c region, B2 region, and/or B2c region of the LAMP product.

12. A method according to claim 10, wherein a part of the target sequence overlaps with a part of the sequence of F2 region, F2c region, B2 region, and/or B2c region of the LAMP product.

13. A method according to claim 1, wherein the presence or absence of the nucleic acid for measurement hybridized to the probe nucleic acid is detected based on a fluorescent label.

14. A method according to claim 2, wherein the presence or absence of the nucleic acid for measurement hybridized to the probe nucleic acid is detected based on a fluorescent label.

15. A method according to claim 1, wherein the presence or absence of the nucleic acid for measurement hybridized to the probe nucleic acid is detected electrically using a double-strand specific intercalator.

16. A method according to claim 2, wherein the presence or absence of the nucleic acid for measurement hybridized to the probe nucleic acid is detected electrically using a double-strand specific intercalator.

17. A LAMP product amplified by LAMP method for detection of the target sequence, wherein the target sequence is inserted between F1 region and F2 region, between F2c region and F1c region, between B1 region and B2 region, and/or between B2c region and B1c region of the LAMP product.

18. A LAMP product according to claim 17, wherein a part of the target sequence overlaps with a part of the sequence of F2 region, F2c region, B2 region, and/or B2c region of the LAMP product.