Methods for detecting target nucleic acid sequences

• US 6,326,145 B1
• Filed: 11/25/1998
• Issued: 12/04/2001
• Est. Priority Date: 06/13/1998
• Status: Expired due to Term

• Current Assignee: Zeneca Incorporated (Astrazeneca PLC)
• Original Assignee: Zeneca Incorporated (Astrazeneca PLC)
• Inventors: Gibson, Neil James, Whitcombe, David Mark, Theaker, Jane, Little, Stephen
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/200,232
• Time in Patent Office: 1,105 Days
• Field of Search: 435/91.2, 435/6, 435/91.1, 536/24.3, 536/23.1, 536/25.4, 536/26.42
• US Class Current: 435/5
• 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 polymerization thereof, under conditions such that the template binding region of the primer will hybridize 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 hybridize to a sequence in the primer extension product corresponding to the target nucleic acid, and wherein any such target specific hybridization 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 method for the detection of a target nucleic acid, which method comprises:

(a) bringing together in a nucleic acid hybridization medium (i) nucleic acid from a sample, said nucleic acid comprising a template nucleic acid sequence and a target nucleic acid sequence, and (ii) a tailed nucleic acid primer comprising, in 5′

to 3′

order, a template binding region and a tail comprising a linker and a target binding region, in the presence of appropriate nucleoside triphosphates and an agent for polymerization thereof, under conditions such that the template binding region of the primer will hybridize to a complementary sequence in the template nucleic acid and be extended to form a primer extension product, the tail of the nucleic acid primer remaining uncopied during amplification, and

(b) separating any said product from the template, whereupon the target binding region in the tail of the primer will hybridize to a sequence in the primer extension product complementary to the target nucleic acid, and wherein any such target specific hybridization is detected via a detectable change in a 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.

2. A method as claimed in claim 1 wherein the tailed nucleic acid primer is used as an amplification primer in an amplification system.

3. A method as claimed in claim 2 wherein the amplification system is the polymerase chain reaction (PCR).

4. A method as claimed in claim 2 or 3 wherein the linker in the tail comprises a blocking moiety to prevent copying of the tail.

5. A method as claimed in claim 4 wherein the tail of the nucleic acid primer comprises a non-copiable species.

6. A method as claimed in claim 1, 2 or 3 wherein hybridisation of the tailed primer to template nucleic acid is performed at a stringency so as to allow primer extension on related template sequences.

7. A method as claimed in claim 6 wherein the related template sequences are human leukocyte antigen (HLA) sequences.

8. A method as claimed in claim 1, 2 or 3 wherein hybridisation of the template binding region and/or target binding region of the primer to a complementary sequence is allele specific.

9. A method as claimed in claim 1, 2 or 3 and using more than one nucleic acid primer for the detection of more than one target nucleic acid sequence.