Methods of nucleic acid analysis

• US 20060223064A1
• Filed: 06/24/2004
• Published: 10/05/2006
• Est. Priority Date: 06/27/2003
• Status: Abandoned Application

• Current Assignee: Matforsk AS
• Original Assignee: Matforsk AS
• Inventors: Rudi, Knut
• Application Number:
  US10/561,710
• Publication Number:
  US 20060223064A1
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The present invention relates to a method of characterising a target base in a sample nucleic acid, which method comprises: (a) contacting the sample nucleic acid with an oligonucleotide primer under conditions which allow hybridisation of the oligonucleotide to the sample nucleic acid, said oligonucleotide primer being labelled with a fluorophore; (b) contacting the sample nucleic acid with a deoxynucleotide or dideoxynucleotide which is labelled with a fluorophore, under conditions which allow extension of the oligonucleotide primer through incorporation of the labelled nucleotide; and (c) measuring the fluorescence emitted by one or both of the fluorophores.

Claims

1. A method of characterising a target base in a sample nucleic acid, which method comprises:

(a) contacting the sample nucleic acid with an oligonucleotide primer under conditions which allow hybridisation of the oligonucleotide to the sample nucleic acid, said oligonucleotide primer being labelled with a fluorophore;

(b) contacting the sample nucleic acid with a deoxynucleotide or dideoxynucleotide which is labelled with a fluorophore, under conditions which allow extension of the oligonucleotide primer through incorporation of the labelled nucleotide; and

(c) measuring the fluorescence emitted by one or both of the fluorophores.

2. A method according to claim 1, wherein one fluorophore can act as a donor and the other fluorophore can act as an acceptor.

3. A method according to claim 1 wherein the oligonucleotide primer fluorophore acts as a donor and the nucleotide fluorophore acts as an acceptor.

4. A method according to claim 1 wherein the oligonucleotide primer fluorophore acts as an acceptor and the nucleotide fluorophore acts as a donor.

5. A method according to claim 2 wherein fluorescence resonance energy transfer can take place between the donor and the acceptor fluorophore when the primer is extended by incorporation of the labelled nucleotide.

6. A method according to claim 1 wherein step b) further comprises contacting the sample with a DNA polymerase and carrying out a thermo-cycling reaction.

7. A method according to claim 1 wherein step c) comprises irradiating the sample nucleic acid and measuring the fluorescence emitted by one or both of the fluorophores.

8. A method according to claim 1 wherein the fluorescence emitted by the fluorophore of the oligonucleotide primer is recorded.

9. A method according to claim 1 wherein the fluorescence emitted by the fluorophore of the deoxynucleotide or dideoxynucleotide is recorded.

10. A method according to claim 1 wherein the primer is designed such that the 3′

end of the primer hybridises immediately upstream of the target base.

11. A method according to claim 1 wherein the labelled nucleotide is a dideoxynucleotide.

12. A method according to claim 1 wherein a plurality of target bases are characterised.

13. A method according to claim 1 wherein only one species of labelled primer is used in step a) and only one species of labelled nucleotide is used in step b).

14. A method according to claim 12 wherein one species of labelled primer and a plurality of different species of labelled nucleotides are used.

15. A method according to claim 14 wherein each species of nucleotide is labelled with a different type of fluorophore.

16. A method according to claim 12 wherein a plurality of different species of labelled primers and one species of labelled nucleotide are used.

17. A method according to claim 16 wherein each species of primer is labelled with a different type of fluorophore.

18. A method according to claim 1 wherein the fluorescence emission maxima of the two fluorophores are at least 15 nm apart.

19. A method according to claim 18 wherein the fluorescence emission maxima of the two fluorophores are at least 30 m apart.

20. A method according to claim 1 wherein the wavelength of the light used for irradiation is such that the light is only efficiently absorbed by the donor and direct excitation of the acceptor is negligible.

21. A kit for use in a method according to claim 1 which kit comprises:

a) an oligonucleotide primer labelled with a fluorophore;

b) a deoxynucleotide or dideoxynucleotide labelled with a fluorophore;

and optionally;

c) a polymerase.