Method for detecting a target nucleic acid sequence

• US 7,919,252 B2
• Filed: 12/22/2008
• Issued: 04/05/2011
• 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
• Primary Examiner(s): Whisenant; Ethan
• Application Number:
  US12/341,295
• Publication Number:
  US 20090104621A1
• Time in Patent Office: 834 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• 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 sequence portion of a nucleic acid comprising:

providing for measurement a stem-and-loop structured nucleic acid, which comprises a double-stranded portion being formed by hybridization of complementary sequence portions on a nucleic acid strand; and

a looped single-stranded portion containing a target sequence portion which connects the two complementary sequence portions;

providing an immobilized probe nucleic acid having a sequence complementary to the target sequence portion which is immobilized at one end to a solid substrate surface;

reacting the stem-and-looped nucleic acid provided for measurement with the immobilized probe nucleic acid for a time and under conditions sufficient to specifically hybridize the target sequence portion of the stem-and-loop nucleic acid provided for measurement to the immobilized probe nucleic acid; and

detecting presence or absence of hybridization between the stem-and-loop structured nucleic acid provided for measurement and the immobilized probe nucleic acid, and detecting the target nucleic acid sequence when hybridization occurs;

wherein 5′

to 3′

sequence orientations of both the immobilized 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 are hybridized.

2. The method according to claim 1, wherein the stem-and-loop nucleic acid for measurement and the immobilized probe nucleic acid are DNA.

3. The method according to claim 1, wherein the immobilized probe nucleic acid is a component of a DNA chip.

4. The method according to claim 1, wherein the stem-and-loop structured nucleic acid for measurement is a LAMP product amplified by a LAMP method, wherein the LAMP product comprises at least one part selected from the group consisting of a first part, a second part, a third part and a forth part of the LAMP product, the first part of the LAMP product comprising F1c, F2c and F1 arranged in this order, the second part of the LAMP product comprising F1c, F2 and F1 arranged in this order, the third part of the LAMP product comprising B1c, B2c and B1 arranged in this order, and the forth part of the LAMP product comprising B1c, B2 and B1 arranged in this order.

5. The method according to claim 4, 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.

6. A method according to claim 5, wherein a part of the target sequence overlaps with a part of the sequence of the F2 region, F2c region, B2region, and/or B2c region of the LAMP product.

7. The method according to claim 1, wherein the presence or absence of the stem-and-loop structured nucleic acid for measurement hybridized to the immobilized probe nucleic acid is detected based on a fluorescent label.

8. The method according to claim 1, wherein the presence or absence of the stem-and-loop structured nucleic acid for measurement hybridized to the immobilized probe nucleic acid is detected electrically using a double-strand specific intercalator.

9. The method of claim 1, wherein the immobilized probe nucleic acid is a component of a current detection type DNA chip.

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

providing for measurement a stem-and-loop structured nucleic acid produced by LAMP (loop mediated isothermal amplification) containing a target nucleic acid sequence in a single-stranded loop portion of said nucleic acid,

contacting for a time and under conditions suitable for hybridization said stem-and-loop structured nucleic acid with an immobilized probe that binds to the target nucleic acid sequence located in a single-stranded loop portion of the stem-and-loop nucleic acid, and

detecting the target nucleic acid sequence when hybridization occurs;

wherein 5′

to 3′

sequence orientations of both the immobilized 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 are hybridized.