Methods and primers for detecting target nucleic acid sequences

• US 9,273,350 B2
• Filed: 06/08/2004
• Issued: 03/01/2016
• Est. Priority Date: 06/13/1998
• Status: Expired due to Fees

• Current Assignee: Qiagen Manchester Limited (Qiagen NV)
• Original Assignee: Qiagen Manchester Limited (Qiagen NV)
• Inventors: Whitcombe, David Mark, Theaker, Jane, Gibson, Neil James, Little, Stephen
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US10/862,580
• Publication Number:
  US 20050164219A1
• Time in Patent Office: 4,284 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/6.12, 435/4, 536/23.1, 536/24.3, 536/25.4, 536/26.43, 536/23.4
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/119 Fluorescence polarisation
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide

Abstract

A method for the detection of a target nucleic acid, which method comprises contacting template nucleic acid from a sample with (i) a signalling system and (ii) a tailed nucleic acid primer having a template binding region and the tail comprising a linker and a target binding region, in the presence of appropriate nucleoside triphosphates and an agent for polymerisation thereof, under conditions such that the template binding region of the primer will hybridise to a complementary sequence in the template nucleic acid and be extended to form a primer extension product, separating any such product from the template whereupon the target binding region in the tail of the primer will hybridise to a sequence in the primer extension product corresponding to the target nucleic acid, and wherein any such target specific hybridisation causes a detectable change in the signalling system, such that the presence or absence of the target nucleic acid in the sample is detected by reference to the presence or absence of a detectable change in the signalling system.

Claims

1. A detection system for detection of a template nucleic acid

wherein the detection system comprises a primer which comprises, in 3′

to 5′

order;

(i) a first binding region and

(ii) a tail comprising a linker and a second binding region,

wherein the tail further comprises at least two regions of internal hybridization;

wherein said tail is not copiable;

wherein the linker comprises a blocking group;

wherein said template nucleic acid to be detected has a first target region and a second target region;

wherein said first binding region is complementary to and binds to said first target region on said nucleic acid to be detected;

wherein said second binding region has the same sequence as the second target region and binds to the complement of said second target region;

wherein said primer is extendible when bound to said first target region to produce a primer extension product which comprises a sequence complementary to said second target region sequence and which primer extension product binds to said second binding region;

wherein the detection system further comprises an integral signaling system provided on the primer molecule that indicates binding of said primer extension product to said second binding region; and

wherein in the absence of the primer extension product or the presence of the primer extension product bound to the template nucleic acid, the at least two internal hybridization regions hybridize with each other to stabilize said integral signaling system in a given position,

wherein in the presence of the primer extension product, wherein the primer extension product is not bound to the template nucleic acid, the second binding region of the primer hybridizes to the primer extension product and stabilizes the integral signaling system in a given position.

2. The detection system of claim 1, which further comprises said primer extension product which comprises a sequence complementary to said second target region sequence and which binds to said second binding region.

3. The detection system of claim 1, which further comprises said template nucleic acid having a first target region and a second target region.

4. The detection system of claim 1 wherein the tail carries an intercalating dye.

5. The detection system of claim 1 wherein the tail comprises a fluorophore for the detection of target binding by fluorescence polarization.

6. The detection system of claim 1 which further comprises a separate species comprising at least one component of an integral signaling system, wherein said component is releasably attached to the tail.

7. The detection system of claim 6 wherein the signalling system comprises energy transfer between fluorophore and quencher species.

8. The detection system of claim 1 wherein the tail acts as a quencher species.

9. The detection system of claim 1 wherein the tail comprises a self-complementary stem duplex having a fluorophore quenched by a quencher species, and wherein the fluorophore becomes unquenched when the stem duplex is disrupted.

10. The detection system of claim 1 which further comprises a capture region which hybridizes to complementary sequence on a solid phase.

11. The detection system claim 1 wherein the second binding region is at least 6 nucleotides long.

12. The detection system of claim 1 wherein the second binding region is 6-50 nucleotides long.

13. The detection system of claim 1, wherein one of the at least two regions of internal hybridization is 3′

of the second binding region and at least one of the at least two regions of internal hybridization is 5′

of the second binding region.