Detection method

• US 20040072247A1
• Filed: 10/08/2003
• Published: 04/15/2004
• Est. Priority Date: 10/03/2000
• Status: Active Grant

• Current Assignee: Id+Plus Limited
• Original Assignee: Id+Plus Limited
• Inventors: Pfistershammer, Josef
• Granted Patent: US 7,455,968 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

A method of identifying a base at a target position in a single-stranded sample DNA sequence, the method characterised by the steps of: (i) providing an oligonucleotide probe which is capable of hybridising to the sample DNA sequence such that the 3′ nucleotide of the probe overlaps the target position, the probe being blocked at the 3′ end to prevent extension by DNA polymerase but permitting 3′-5′ exonuclease cleavage; (ii) incubating the sample DNA sequence with the probe in the presence of (a) a composition having 3′-5′ exonuclease activity and (b) nucleotide triphosphates, under conditions that allow hybridisation of the probe to the sample DNA sequence; and(iii) detecting, directly or indirectly, cleavage of the 3′ nucleotide of the probe.

Claims

1. A method of identifying a base at a target position in a single-stranded sample DNA sequence characterised in that the method comprises:

(i) providing a DNA probe which is capable of hybridising to said sample DNA sequence such that the 3′

nucleotide of the probe overlaps the target position;

(ii) incubating said sample DNA sequence with said probe in the presence of (a) a composition having 3′

-5′

exonuclease activity and (b) nucleotide triphosphates, under conditions that allow hybridisation of the probe to the sample DNA sequence; and

(iii) detecting, directly or indirectly, cleavage of the 3′

nucleotide of the probe.

2. A method according to claim 1, characterised in that the 3′

nucleotide of the probe is terminally blocked.

3. A method according to claim 1 or 2, characterised in that the probe comprises;

a) a fluorescent moiety and a quencher moiety, the fluorescent moiety or the quencher moiety, but not both, being attached to the 3′

nucleotide of the probe, and step (iii) comprises detecting an increase in fluorescence resulting from the direct cleavage of the fluorescently labelled probe and/or

b) a probe according to claim 1 which includes a 3′

blocked terminal mismatch nucletide and an additional probe which is fluorescently labelled as in claim 3A and able to simultaneously hybridise with the sample strand of DNA within approximately 500 nucleotides of the blocked, mismatch probe, and step iii) consists of detecting indirectly the cleavage of the first probe through the action of the 5′

-3′

exonuclease activity of a DNA polymerase on the downstream fluorescent probe.

4. A method according to claim 1 or 2, characterised in that step (iii) comprises detecting the formation of pyrophosphate produced by extension of the cleaved probe by a DNA polymerase.

5. A method according to anyone of claims 1, 2 or 4, characterised in that the detection of pyrophosphate comprises contacting the pyrophosphate with a means for converting pyrophosphate to adenosine triphosphate (ATP) and detecting the presence of ATP.

6. A method according to any one of claims 1, 2, 4 or 5, characterised in that the ATP is detected by contacting the ATP with means for hydrolysing ATP with concomitant production of light.

7. A method according to claim 6, characterised in that the light is detected by optical means.

8. A method according to any one of the preceding claims, characterised in that two or more probes are provided and which hybridise to the same location in the sample DNA sequence and which have a different base at the 3′

end.

9. A method according to any one of the preceding claims, characterised in that the probe or probes are immobilised to a solid phase.

10. A method according to any one of the preceding claims, characterised in that a plurality of probes are provided for the identification of a plurality of target positions, each different probe being provided in a discrete location on the solid phase.

11. A method according to any one of the preceding claims, characterised in that each probe is provided at a plurality of positions on the solid phase.

12. A method according to any one of the preceding claims, characterised in that the probes are provided as a discrete array on a solid phase and the array is moved relative to the means for detecting the cleavage of the 3′

nucleotide of any probe in a continuous or semi-continuous manner.

17. An apparatus for use in the method of any one of the preceding claims, characterised in that the apparatus comprises:

(i) A detection device for detecting an optical change at one or more discrete positions on a film, said film being moved relative to the detection device in a unidirectional continuous or semi-continuous manner;

(ii) A device for moving the film relative to the detection device; and

(iii) One or more devices for applying nucleotide extension primers and/or reagents at discrete positions on said film prior to, or concomitant with, the detection of an optical change at said positions.

13. The use of a DNA probe in a method of identifying a base at a target position in a single-stranded sample DNA sequence characterised in that the probe is capable of hybridising to said sample DNA sequence such that the 3′

nucleotide of the probe overlaps the target position, the 3′

nucleotide being terminally blocked so as to prevent primer extension by a DNA polymerase enzyme.

14. A kit comprising a plurality of DNA probes for use in the identification of a nucleotide base at a plurality of target positions in a single stranded DNA sample, characterised in that each probe is capable of hybridising to said sample DNA sequence such that the 3′

nucleotide of the probe overlaps a target position, the 3′

nucleotide being terminally blocked so as to prevent primer extension by a DNA polymerase enzyme of the uncleaved probe.

15. A kit according to claim 14, characterised in that the probes are provided in groups, each group comprising two or more probes which have a different base at the 3′

end and which hybridise to the same location in the sample DNA sequence.

16. A kit according to claim 14 or 15, characterised in that the kit comprises a composition having a 3′

-5′

exonuclease activity; and

optionally a DNA polymerase enzyme and/or a reagent capable of converting the formation of pyrophosphate into an optically detectable signal.