Detection method
AssigneeID+PLUS Ltd.
InventorJosef Pfistershammer
Life of this patent
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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.
Claims17
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.
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.
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.
A method according to any one of the preceding claims, characterised in that the probe or probes are immobilised to a solid phase.
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.
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.
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.
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.
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.
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.
Dependencies are inferred from the claim text.