Nucleic acid sequence identification

• US 7,001,721 B1
• Filed: 05/20/1999
• Issued: 02/21/2006
• Est. Priority Date: 05/20/1998
• Status: Expired due to Fees

• Current Assignee: Renishaw Diagnostics Limited (Renishaw PLC)
• Original Assignee: University of Strathclyde
• Inventors: Graham, Duncan, Smith, William Ewen, Whitcombe, David Mark
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Assistant Examiner(s): Chakrabarti, Arun
• Application Number:
  US09/700,732
• Time in Patent Office: 2,469 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/5, 536/24.3, 935/6, 935/77, 935/78, 436/518, 436/164, 436/173, 436/525, 436/801, 436/805
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer
• CPC Class Codes: G01N 21/658 enhancement Raman, e.g. surface plasmons

Abstract

Disclosed are methods for determining the presence or absence of a target nucleic acid (e.g. DNA) sequence in a sample nucleic acid, the method comprising: (a) exposing the sample to a detection agent comprising a colloid metal surface associated with a SER(R)S active species (SAS) such as an azo dye and with a target binding species (TBS) which may be PNA which is complementary to the target, and (b) observing the sample agent mixture using SER(R)S to detect any surface enhancement of the label wherein the binding of the TBS to the target sequence causes surface enhancement SAS. The detection agent may be exposed to the sample in step (a) as two or more separate components and will generally comprise a first agent and a second agent each having a different TBS, each TBS being capable of binding to the target sequence, and wherein the binding of the first and second TBS to the target sequence brings a metal surface associated with each TBS into proximity thereby causing surface enhancement of an SAS associated with one or both of the metal surfaces.

Claims

1. A method for determining the presence or absence of a target nucleic acid sequence in a sample nucleic acid, the method comprising:

(a) exposing the sample to a detection agent comprising at least two separate components, including a first agent having a metal surface associated with a first target binding species (TBS) and a second agent having a metal surface associated with a second TBS, different from said first TBS, at least one of said metal surfaces being associated with a SER(R)S-active species (SAS), each of said first and second TBS being effective to bind to the target sequence, and wherein the binding of the first and second TBS to the target sequence causes aggregation of the metal surfaces associated with said TBS, thereby causing surface enhancement of a SAS associated with one or both of the metal surfaces, said metal surfaces being ineffective to cause surface enhancement in the form in which they are present in the detection agent to which said sample is exposed, and aggregation of said metal SER(R)S surface being dependent on the presence of said target nucleic acid in said sample; and

,

(b) observing the sample/agent mixture using SER(R)S to detect any said surface enhancement.

2. The method as claimed in claim 1 wherein each component of said detection agent comprises monodisperse unaggregated colloidal metal particles associated with a TBS comprising a nucleic acid or nucleic acid analog which is complementary to all or part of the target sequence.

3. The method as claimed in claim 2 wherein the TBS comprises propargyl amino modified nucleic acid or peptide nucleic acid.

4. The method as claimed in claim 2 wherein there are up to 20 TBS per metal colloid particle.

5. The method as claimed in claim 1 wherein a surface seeking group (SSG) is used to promote chemisorption of at least one of the SAS and TBS to the metal surface.

6. The method as claimed in claim 5 wherein the SSG comprises a triazole group.

7. The method as claimed in claim 5 wherein the SSG is modified with a dye which is a SAS.

8. The method as claimed in claim 7 wherein the modified SSG is an azobenzotriazole.

9. The method as claimed in claim 7 wherein the modified SSG is used to associate the TBS to the metal surface.

10. The method as claimed in claim 9 wherein the modified SSG is conjugated to the TBS via a linker group.

11. The method as claimed in claim 1 wherein the SAS is present in an amount of up to 100 fold excess over the TBS.

12. A method as claimed in claim 1 wherein more than one target sequence is determined using detection agent components having distinguishable SAS.

13. A method as claimed in claim 12 wherein the target sequences share sequence identity, and wherein a common first agent is used in conjunction with specific distinguishable second agents which can discriminate between the remainder of the target sequences.

14. A method for detecting the presence of, or selecting, or identifying, or phylogenetically classifying, an organism,, the method comprising use of a method as claimed in claim 1 wherein the target nucleic acid sequence is associated with that organism.

15. A method for diagnosing a disease, the method comprising use of a method as claimed in claim 1 wherein the target nucleic acid sequence is associated with that disease.

16. A method for isolating a nucleic acid encoding a specific gene, the method comprising use of a method as claimed in claim 1 wherein the target sequence corresponds to a sequence associated with, or within, that gene.

17. The method as claimed in claim 6 wherein said triazole group is the benzotriazole group.