Methods and primers for detecting target nucleic acid sequences

• US 20050164219A1
• Filed: 06/08/2004
• Published: 07/28/2005
• Est. Priority Date: 06/13/1998
• Status: Active Grant

• Current Assignee: Qiagen Manchester Limited (Qiagen NV)
• Original Assignee: DxS Limited (Qiagen NV)
• Inventors: Gibson, Neil James, Theaker, Jane, Little, Stephen, Whitcombe, David Mark
• Granted Patent: US 9,273,350 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 diagnostic primer comprising in 5′

to 3′

order

(i) a template binding region and

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

said template binding region being complementary to a template nucleic acid sequence of a sample nucleic acid, said sample nucleic acid additionally comprising a target nucleic acid sequence, and said primer being extendable when hybridized to said template nucleic acid sequence in the presence of appropriate nucleoside triphosphates and an agent for polymerization thereof to define a primer extension product, said tail not capable of being copied,

and wherein the target binding region is hybridizable with a complementary sequence in said primer extension product corresponding to the target nucleic acid, and the complementary sequence is less than 200 base pairs from the template binding region.

3. A primer as claimed in claim 1 or 2 wherein the linker comprises a blocking moiety which prevents polymerase mediated copying of the primer tail.

4. A primer as claimed in claim 1 or 2 and further comprising at least one component of an integral signaling system to indicate hybridisation of the target binding region to a complementary sequence in a primer extension product of the primer.

5. A primer as claimed in claim 4 wherein the primer tail carries an intercalating dye.

6. A primer as claimed in claim 4 wherein the primer tail comprises a fluorophore for the detection of target binding by fluorescence polarisation.

7. A diagnostic primer as claimed in claim 4 and further having a separate species comprising at least one component of an integral signaling system, wherein said component is releasably attached to the primer tail.

8. A primer as claimed in claim 7 wherein the signalling system comprises energy transfer between fluorophore and quencher species.

9. A primer as claimed in claim 4 wherein the primer tail acts as a quencher species.

10. A primer as claimed in claim 3 wherein the primer tail includes one or more regions of internal hybridisation to stabilise one or more component(s) of the signaling system in a given position.

11. A primer as claimed in claim 10 wherein the primer 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.

12. A primer as claimed in claim 1 or 2 which further comprises a capture region which hybridises to complementary sequence on a solid phase.

13. A kit which comprises at least one primer as claimed in claim 1 or 2 together with packaging and instructions for use.

2. A diagnostic primer comprising in 5′

to 3′

order

(i) a template binding region and

(ii) a tail comprising a linker and a target binding regions

said template binding region being complementary to a template nucleic acid sequence of a sample nucleic acid, said sample nucleic acid additionally comprising a target nucleic acid sequence, and said primer being extendable when hybridized to said template nucleic acid sequence in the presence of appropriate nucleoside triphosphates and an agent for polymerization thereof to define a primer extension product, said tail not capable of being copied,

and wherein the target binding region is hybridizable with a complementary sequence in said primer extension product corresponding to the target nucleic acid.