WO2007045890A1

WO2007045890A1 - Probe

Info

Publication number: WO2007045890A1
Application number: PCT/GB2006/003904
Authority: WO
Inventors: Robert Powell
Original assignee: Primerdesign Ltd
Priority date: 2005-10-21
Filing date: 2006-10-20
Publication date: 2007-04-26

Abstract

The present invention relates, in one aspect, to a nucleic acid probe for monitoring or detecting a target nucleic acid in a sample, wherein said probe: (i) comprises a reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule; (ii) has a substantially closed structure in its unhybridised state such that the reporter is substantially quenched; (iii) is modified at the 3' terminus to prevent extension by a nucleic acid polymerase; and (iv) is or is capable of being substantially hydrolysed bythe nucleic acid polymerase, and wherein the nucleic acid probeJias: (i) a target annealing temperature of from about 64°C to about 75°C; and (ii) a melting temperature of from about 45°C to about 58°C.

Description

PROBE

FIELD OF THE INVENTION

The invention relates to nucleic acid probe that includes a reporter molecule and a quencher molecule. The invention also relates to inter alia the use of such probes in nucleic acid amplification, reactions - such as -the polymerase- chain reaction .(PCR) and Real-Time PCR.

INTRODUCTION

Reporter molecules and quenchers have been incorporated into oligonucleotide probes to monitor biological events, based on the reporter molecule and quencher being separated by some mechanism to increase .reporter emission, or brought into proximity to reduce reporter activity.

One particularly -important application for the use of oligonucleotide probes is in nucleic acid amplification reactions - sueh as PCR. Nucleic acid amplification reactions and in particular -PCR has -become a research tool^~of major importance with applications in gene expression analysis, cloning, DNA sequencing, genetic mapping and drug discovery. Real time BCR^" is simply a PGR reaction_^ wherein the amplification of^~nucleic acid can be monitored^" in "real time". Approaches. to- real time monitoring of PCR fallTh to two major categories. Firstly tibat of intercalating dyes wherein ^ dye which fluoresces^~uponJntercalation-with double -stranded DNA (e.grΕώiidium^'bromide)-is used-to indicate the relative volumes of DNA -present in the reaction ~vesseL Alternatively, and coneerningjhis invention, oligonucleotide probes which include a fluorescent reporter molecule and quencher- are used.

Probes containing a reporter molecule and quencher have been developed for hybridisation assays whereby the probe forms a hair-pin structure i.e. the probe folds on itself to form a loop such that the reporter and quencher on either end of the probe are in to close proximity (also referred to as Molecular beacons. S. Tyagi and F.R. Kramer, Nat. Biotechnol. 1996, 14, 49). In the presence of a complimentary polynucleotide sequence the hair-pin structure of the probe melts and the probe binds to the complementary target sequence. The quencher molecule therefore is spatially separated from the fluorescent reporter by the length of the probe. As a result an increase in fluorescence emission is detectable. Probes including a hair-pin structure have the disadvantage that the quencher and reporter molecules are removed from proximity to each other only by the conformational change in the probe structure upon hybridisation to the target polynucleotide sequence. Thus, the reporter remains quenched to a large extent and any increase in fluorescent emission upon- such a conformational change is limited in its magnitude.

An alternate probe based approach- has been to use oligonucleotide probes with a fluorescent reporter molecule and quencher incorporated: The said quencher being in sufficient proximity to the reporter molecule to significantly reduce fluorescence emission (see-US 5,210,015 - also referred to as the Taq-Man-approach). The- probe is designed to bind to a target polynucleotide sequence. Upon amplification, the probe is hydrolysed by the 5' to 3' exonucϊease activity of the polymerase thus separating tire quencher from the reporter molecule. Since the probe and quencher are separated completely a larger increase in fluorescence is detectable upon amplification of the target polynucleotide sequence. However such an approach_has the disadvantage that the quenching of the fluorescent reporter molecule by the quencher molecule is dependent upon the proximity of the quencher to the reporter molecule. As the .reporter molecule -and^' quencher are sepaτated_by a- number of nucleotide bases -equal" to the length Of ^"the prebe,. the efficiency of- the quencher 4o inhibit fluorescence emission- is limited. Hence, a high level of "background fluorescence" is-always present when using such an assay. This reduces the signal to noise ratio that can be -achieved- with the assay -and-relduces- the- sensitivity of -said •assay.

Kong D et al . -Chem Common (C-amb). 2002 Apr 21;(8):854-5 report a modified molecular beacon combing the properties of a Taq-Man probe. Said probe includes a fluorescent reporter molecule on the 5' terminus and a quencher molecule capable of quenching the fluorescent emission of said reporter molecule on the 3' terminus. The probe is modified by the addition of 6 nucleic acid bases to the 3' end complementary to the 6 nucleotide bases at the 5' end such that the probe will form a hairpin loop structure similar to that of a molecular beacon. Upon hybridisation to a target polynucleotide sequence the said probe undergoes a conformational change to a linear conformation. Upon amplification of the target polynucleotide sequence a proportion of probe is hydrolysed by the 5' to 3' endonuclease activity of the polymerase thus separating the reporter molecule from said probe resulting in an increase in fluorescent emission. The annealing temperature of the probe described therein is 63 ⁰C and the melting temperature is 62.5 ⁰C. The probe design is not optimised to allow complete hydrolysis of the probe during amplification of the target polynucleotide sequence. Moreover, in addition -to standard PCR cycling conditions an additional "hybridisation .step" is required thus complicating-ihe use of the assay and slowing- the rate at- which experiments can be performed.

A need currently exists for effective oligonucleotide probes containing a reporter -molecule and quencher molecule for use in hybridisation assays with optimal quenching and hydrolysis properties.

Thejpresent invention seeks to overcome the problems-θf the prior art.

SUMMARY ΘF THE INVEOTION

The present invention is based, in part^ on the surprising finding that optimising, for.

-example, the annealing- and- melting temperatures of nucleic acidηprobes containing inter alia a reporter molecule and^" quencher molecule results in nucleic acid" probes with a number of beneficial and even unexpected properties. In particular, when the

- nucleic acid probes are used in an amplification reaction {e.g. PCR), the probes have a number of -beneficial and even unexpected -properties. Accordingly, the present invention advantageously provides a- nucleic add probe which- when -unhybridised, exhibits-excellent self-quenching properties due -to the proximity of the quencher with the reporter molecule. In addition the probe exhibits a conformational change in structure upon hybridisation with a complementary target polynucleotide sequence resulting in a reduction in the proximity of the quencher and reporter molecule, and a subsequent substantial increase in fluorescence emission. Moreover, said probe has optimal properties to allow the substantial (eg. complete) hydrolysis of said probe by the activity (eg. the 5' to 3' exonuclease activity) of a polymerase, thus separating the reporter molecule from the quencher molecule upon amplification of the target polynucleotide sequence, leading to maximum increase in 5 detectable fluorescence.

Surprisingly, the nucleic acid probe described herein is also more sensitive than the probes σf the prior art - such as TaqMan probes and Molecular Beacons - when used in an amplification reaction-(e.g. PCR). lθ-

According _to the- data presented" herein and the extensive research that has been conducted, it is predicted that the probes described in Kong et al. {supra) would not perform comparably^ with.- the. present invention. Most significantly- the probes described therein would not be substantially melted at the probe annealing

15 temperature and therefore binding and subsequent hydrolysis^r would- be much less efficient than with the-probes of the present invention.

SUMMARY ASPECTS OF THE PRESENT INVENTION

2α In a first aspect, there is provided^' a nucleic acid probe for monitoring or detecting a- target nucleic acid-in-a sample, wherein said probe: (i) comprises a reporter molecule" and a quencher molecule capable -of quenching the reporter molecule; (ii)--has a substantially closed structure in its unhybridised state such ϊhairthe reporter is substantially quenched;_(iii)-is modified at the 3' terminus to prevent extension by a

25 nucleic acid polymerase; .and (iv)3s or is .capable of being"substantially^"hydroiysed by tfee nucleic -acid polymerase^ and wherem=ihe nucleic acid probe has: (a) a-target annealing temperature -of from about J54 ⁰C tσ about 75 ⁰C; and (b) a -melting temperature of from about 45 ⁰C to about 58 ⁰C.

30 Suitably, the probe has a substantially closed structure in its unhybridised state at the amplification reaction {eg. PCR) annealing temperature such that the reporter is substantially quenched. In a second aspect, there is provided a method for monitoring or detecting a target nucleic acid in a sample comprising the use of a nucleic acid probe according to the first aspect of the present invention.

5 In a third aspect, there is provided a method for monitoring or detecting a target nucleic acid in an amplification reaction comprising the steps of: (a) contacting a sample of nucleic acid with a nucleic acid probe according to the first aspect of the present invention and one -or -more nucleic acid primers, wherein the amplification reaction annealing temperature is lower than the melting-temperature of the probe; (b) 0 amplifying the -target nucleic acid; and (c) following and/or during the amplification reaction measuring the reporter activity.

In a fourth aspect, there is provided a method for- preparing a nucleic acid probe for monitoring or detecting a target nucleic acid in a sample, wherein said probe: (-i) 5 comprises a reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule; (_ii). has- a substantially closed structure in its unhybridised state such that the reporter is substantially -quenched; (ϊϋ)-is modified at -the 3' terminus to prevent extension by a nucleic acid^'polymerase; and (iv) is or is capable of being substantially hydrolysed by the nucleic acid-polymerase, comprising 0- the steps of: (a) identifying a nucleic acid probe sequence that -can be used to-monitor or detect .a^~targetjiuclelc acid; (b) adjusting the -design-ofihe nucleic acid probersuch- -that-the annealing temperature of said-probe" is from about .64 ⁰C to about 75^~°C; and (c) adjusting the design of the nucleic acid probe such that the melting temperature of said-probe is-from about 45 ⁰C to about 58 ⁰G. S

In a fifth aspect, mere is provided^" the- use of the nucleic acid probe_accer4ing to. the first aspect of the present invention for monitoring or -detecting a target nucleic acid in a sample.

0 In a sixth aspect, there is provided a kit comprising a nucleic acid probe according to the first aspect of the present invention and a nucleic acid polymerase and optionally, one or more primers.

SUVTMATRY EMBODIMENTS OF THE PRESENT INVENTION In one embodiment, the closed structure is a hairpin-loop structure.

In another embodiment, the-proximity of the quencher to the reporter is reduced when the probe is at the target annealing temperature.

In another embodiment,-the reporter is a fluorescent reporter.

In another embodiment, the quencher is a fluorescent quencher.

In- another embodiment, said probe is or is capable -of being substantially Jiydrόlysed by a nucleic acid polymerase having 5' to 3' exonuclease activity.

In another embodiment, the probe is completely hydrolysed.

In another embodiment_^ the probe-comprises one or more additional nucleic hases-at the 5' end of the oligonucleotide.

In another embodiment, the probe comprises one or more additional nucleic bases -at the 3' end of the olirgonucleotide..

In-ansther embodiment; the prøbe- comprises one or more- additional- nucleic bases- at the 5'"arid^"3' ends ofthe oligonucleotide that are complementary to each other.

In ^"another embodiment, the annealing temperature of the probe is higher than the annealing- temperature of the-primer(s)^".

In another embodiment, "the annealing temperature ofthe primers is from about 52-58 ⁰C.

In another embodiment, the read temperature ofthe amplification reaction is from 40- 59 ⁰C. In another embodiment, said probe hybridises to the target sequence 3' relative to the one or more primers.

In another embodiment, the primers are linear DNA -molecules with no internal structure.

In another embodiment, nucleic acid is amplified using PCR.

In another- embodiment, nucleic acid is amplified using Real-time PCR.

In another embodiment, the amplification reaction is .performed at a denaturation temperature of about 95 ⁰C, an annealing temperature of about 50 ⁰C and an extension temperature of about 72 ⁰C.

In another embodiment, the reporter activity is measured during the annealing stage of the amplification Teaction.

DESCRIPTION OF THE FIGURES

Ih some of the Figures,..assays targeting-the-samejregion-of gene-AAAl using identical -primers-are illustrated. The Taqman probe has-a target^" annealing temperature of 68.6 ⁰C. The Molecular Beacomprobe has a target-annealing temperature of=58τl ⁰C and a. hairpin melting temperature of 56,8 ⁰C. The probe labeled "TAQman-5tag" has a target annealing temperature of 68.6 ⁰C and a hairpinτmelting temperature of 58 ⁰C. TheTCR cycling conditions used were 9-5-°C for 8 mins followed by 54- cycles of 95 ⁰C for 1=5 seconds, x ⁰C for 30 s andT2-°C for- 15 s where x- is the annealing temperature indicated in the figure legends.

Figure 1 This figure shows a schematic representation of the present invention in comparison to the "Taq-man" approach and the "Molecular Beacon" approach. Column A illustrates the native conformation of the probe prior to amplification. Column B represents the probe hybridised during the extension phase of the amplification reaction. Column C represents the probe following amplification of the target nucleic acid sequence during amplification. The point at which fluorescence emission is recorded to monitor the progress of amplification of the target nucleic acid sequence in each approach is indicated by the presence of * in each case.

Figure 2

This figure shows a real-time PCR amplification plot comparing the trace obtained from a Taqman style and Molecular Beacon probe. Consistent with Figure 1, the background fluorescence of the Taqman probe is much higher-than that ef the "Beacon. The change in fluorescence (endpoint florescence at 60 cycles - baseline fluorescence) is higher for the Taqman probe. The signal to noise ratio, (endpoint florescence at 60 cycles / baseline fluorescence) is higher for the Beacon probe.

Figure 3 This figure shows the melt curve for a Taqman, Molecular beacon probe and: the present invention. The Taqman probe has no -defined melting curve as it-has-no hair pin structure. The probe of the present invention melts more readily than the Molecular Beacon probe. T probe of the present invention also reaches full dissociation (is fully melted) at a lower temperature than the Beacon Probe.

Figure 4

This figure shows the performance of the present invention- (Taqman-5'Taq) compared to equivalent Taqman and^" Molecular Beacon "Probes at different anneaiing/read temperatures. At 61⁰C the present invention gives a very similar.real- -time PCR trace to÷the_Taqman probe. Thisύs because the hairpin structure-does net form at-6 IZC and- unbound probe -is not optimally quenched at this- temperature. At lower annealing temperatures of, for example, ^"50⁰C and 40.8⁰C the probe of the present invention has much improved quenching properties which is due to the folding of unbound probe into the optimally quenched form. The end point fluorescence of the probe of the present invention remains similar to the Taqman even when hairpin formation is permitted by performing the annealing/read at the lower temperatures. At the lower annealing/read temperatures the quenching of the probe of the present invention is similar to that of the Molecular Beacon. Figure 5

This figure shows the Post PCR melt curves of the Taqman, Molecular Beacon and probe of the present invention. The Taqman probe and the probe of the present invention have no defined melt profile. This indicates that the probe of the present invention has been hydrolysed during the amplification reaction. The Molecular Beacon probe retains its melt, profile after amplification. The. Molecular Beacon probe has not beenJiydrolysed to any significant extent.

Figure 6

Thϊsr figure shows in detaiHhe improvement in sensitivity that can=-be achieved- with the probe of the present invention. The individual data points at each amplification cycle are shown and the data are shown following baseline subtraction. The amplification plot of the probe of the .present invention rises above the-background- level at earlier cycles -than the Taqman or Moleeular Beacon probe. The probe of the present invention is more fiuorøgenic than the Taqman or Molecular Beacon -probe.

Figure 7

This_ figure shows quantitatively the improvement in sensitivity of the present -invention compared to Taqman and Molecular Beacon probes. Relative quantification- in arbitrary units is. obtained by subtraction of the cycle threshold, and calculatingrthe difference in detection (The Δ A_CT method); At 61⁰C the-probe of the present invention is only slightly improved^~over the Taqman probe. At an anneal/read temperature of 50⁰C- the probe of ^"the present invention is much improved over the ^r "detection- obtained with either the Taqman or Molecular ^"Beacon probβr At an annealing/read temperature of 40.8⁰C the probe of the present- invention-is-fπrtiher improved but only marginally.so.

Figure 8 This figure shows a comparison of Taqman, Molecular Beacon probes with the probe of the present invention for a number of different target sequences using a range of hardware platforms ((A) IcyclerlQ; (B) Lightcycler; and (C) Rotorgene). The forward and reverse primers were kept identical within a particular target sequence such that only the probe design varied between comparisons. The probes are shown in Table 1. The probe of the present invention is consistently more sensitive than Taqman and Molecular Beacon probes across a range of platforms and for multiple target sequences. The probe of the present invention gives a consistently higher signal to noise ratio than the Taqman probe and one out of two Molecular Beacon probes.

X)ETAILED DESCRIPTION

PROBE

In one aspect, there is provided a nucleic acid probe for monitoring or detecting a target nucleic-acid in a sample, wherein said probe: (i) comprises a reporter molecule and a quencher molecule capable of quenching the- fluorescence of the reporter molecule; and/or (ii) has a substantially dosed structure in its unhybridised state such that the reporter is substantially quenched; and/or (iii) is modified at the 3' terminus to prevent extension by a nucleic acid polymerase; and/or (iv)Ts or is capable of being substantially hydrσlysed by- the nucleic acid polymerase, and wherein the nucleic-acid probe has: (i) a targelannealing -temperature of from about 64 ⁰C to about 75 ⁰C; and ^" (ii) a -melting, temperature of from about 45 ⁰C to about 58 ⁰C.

As used herein, the term "probe" refers_to an oligonucleotide that forms a duplex structure with a sequence of a target nucleic acid due to complementary base pairing. Typically, -the oligonucleotide probe is in the range of -about 15-60 nucleotides in length. Preferably, .the oligonucleotide probe is in ther range of about 20=60. nucleotides in length, morε--preferably, the oligonucleotide probe -is- in. the^range? of about 25-55 nucleotides in length, more preferably,- the oligonucleotide probe is=in the range of about 25-50 nucleotides in length, more preferably, the oligonucleotide probe is in the range of about 25-45 nucleotides in length, more preferably, the oligonucleotide probe is in the range of about 25-40 nucleotides in length, more preferably, the oligonucleotide probe is in the range of about 25-39 nucleotides in length, more preferably, the oligonucleotide probe is in the range of about 25-38 nucleotides in length, more preferably, the oligonucleotide probe is in the range of about 25-37 nucleotides in length, more preferably, the oligonucleotide probe is about 25-36 nucleotides in length, most preferably, the oligonucleotide probe is about 25- 35 nucleotides in length.

The precise sequence and length of an oligonucleotide probe according to the invention depends in part on the nature of the target polynucleotide to which it binds. The binding location and Jength may be varied to- achieve appropriate annealing and -melting properties for a particular embodiment. Guidance for making such design choices is well known in-the-art.

The probe will comprise a iybridϊsing regioπr preferably consisting of about 1-0 to 50- nucleotides, more preferably about 20 to 40 nucleotides, more preferably about 25 to 35 nucleotides, corresponding (eg. identical to or complementary to the designated nucleic acid) to a region of the target sequence.

An oligonucleotide may be prepared by any suitable method, including, but not limited^" to, cloning and isolation-of sequences using-restriction enzymes and direct chemical synthesis by a method - such as "the phosphotriester method (Meth.- Enzymoi: (1979) 68:90-99; Meth. Enzymol. (1⁼979) 6&109-151); and^' the -diethylphosphoramidite method (Tetrahedron Lett..(1981) 22: 1^'859-1862).

Methods for synthesising labeled oligonucleotides are described in-Agrawal^" and Zamecnik,1990, Nucl. Acids. Res. 18(18):5419-5423; MacMillan and Vetdine, 1990, J.--Org. Chem. 55:5931-5933; Pieles.et al., 1989, Nucl. Acids. Res. 17(22):8967-δ978; -Roger et ah, 1989, Nucl. Acids. Res. 17(19-)r7543-76J-£; and^~Tesler^~et al., Ϊ989, J. Am.- GheHL- -S-OC; 11-1:6966-6976. A review of synthesis methods is provided in Goodchild,-1990, -5røc£w/«gflte Chemistry 1(3): 165- 187.

In a preferred aspect of the present invention there is provided a method for preparing a nucleic acid probe for monitoring or detecting a target nucleic acid in a sample, wherein said probe: (i) comprises a reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule; and/or (ii) has a substantially closed structure in its unhybridised state such that the reporter is substantially quenched; and/or (iii) is modified at the 3' terminus to prevent extension by a nucleic acid polymerase; and/or (iv) is or is capable of being substantially hydrolysed by the nucleic acid polymerase, comprising the steps of: (a) identifying a nucleic acid probe sequence that can be used to monitor or detect a target nucleic

"acid; (b) -adjusting the design^r of the nucleic acid probe such that the annealing temperature of said probe is from about 64 ⁰C to about 75 ⁰C; and (c) adjusting the design of the nucleic acid probe such that the melting temperature of said probe is from about 4-5 *C to about-58 ⁰C.

HYDROLYSIS

The nucleic acid probe when hybridised to Ά complementary polynucleotide will' undergo complete or near complete hydrolysis due to the 5' to 3' exonuclease activity of polymerase. In a preferred embodiment -of the present invention, the nucleic acid probe undergoes 75%..more preferably, .^"80%, more preferably, 8-5%, more preferably, 90%, more preferably, 95%, more preferably, 96%, -more preferably_^ ^"97%, more preferably, 98%, more-preferably, 99% or most- preferably, 1-00% hydrolysis.

Any suitable nucleic acid polymerase having 5' to 3' exonuclease activity and substantially (eg. completely) devoid of 3' to 5' exonuclease activity may be employed. Typically, the nucleic .acid polymerase hydrolases the oligonucleotide

-analytical probe only when the. probe is hybridised to the targetnucleic-acid sequence.

Preferably, there is employed a thermostable nucleic acid DNA polymerase - such as a-thermostablejiucleic a&id DNA-polymerase as=disclbsed4n US 4^889,-818.

Thus the reporter molecule is released into solution and an increase in reporter activityis detected.

The nucleic acid probe described herein is also modified at the 3' terminus to prevent extension by a nucleic acid polymerase. Accordingly, the 3' terminal nucleotide of the oligonucleotide probe is blocked or rendered incapable of extension by a nucleic acid polymerase. Such blocking may be carried out by the attachment of a reporter or quencher molecule to the terminal 3' carbon of the oligonucleotide probe by a linking moiety.

HAIRPIN

5

Advantageously, the nucleic acid probe of the present invention forms secondary structure, which results in -regions of double-stranded DNA. Accordingly, it is a feature of the -nucleic acid probe according that it has a substantially closed structure in its-unhybridisecLstate at the amplification reaction annealing temperature such, that

10- -the reporter is substantially quenched. Secondary structure may be introduced into the single-stranded nucleic acid probes by/ifie -addition of a terminal sequence complementary to the other terminus. The secondary structure formed may involve .the hybridisation "of the 5' and-3^!"ends of the probes to form a "hairpin" structure. The length of the .complementary sequences atjeachrend of the probe must be sufficient to

15 form a stable hairpin secondary structure at the assay annealing .temperature and^: conditions yet not long enough so as to stabilise the hairpin secondary structure so that probe self-hybridisation out competes probe-target hybridisation, rendering- the probe incapable of hybridising to the target-sequence.

20 .The exact sequence of the proberwiil depend=on the -target sequence to be detected -and on -the experimental conditions in_ which tier nucleic -acid probe -is used. Preferably, complementary- terminal regions of about 6, 7, 8 or ^"9 nucleotides- in length- are sufficient to cause^~the formation- of a stable hairpin structure, although more or less complementarity may be desiredLdepending on the reaction conditions. The stability

-25 of- the hairpin secondary structure of-the probe and the stability of the probe-target hybridisation duplex-can be determined empirically.

Accordingly, the probe is typically modified by the addition of nucleic acid bases such that the probe when unhybridised maintains a hair-pin loop conformation. In 30 such a conformation the reporter molecule and quencher molecule are brought in to physical proximity such that the quencher is near enough to the reporter molecule to quench the fluorescence of said reporter molecule. In one embodiment the modification of the probe is achieved by addition of nucleic acid bases to the 3' end of the oligonucleotide of reverse complement to those at the 5' end of the probe. Hence the two ends of the oligonucleotide hybridise to maintain the hair-pin loop conformation of the probe.

In another embodiment the modification of the probe is achieved by addition of nucleic acid bases to the 5' end of the oligonucleotide of reverse complement to those at the 3' end of the probe. Hence the two- ends of the oligonucleotide hybridise to maintain the hair-pin loop conformation of the probe.

In another embodiment the modification of theprobe is achieved by additioirof probe independent nucleic acid bases to both the 3' and 5 'end of the oligonucleotide. Said nucleic acid base additions-are designed- to compliment each other such that the two modifications will hybridise with each other. Hence the^" two .ends of _the oligonucleotide hybridise to maintain the hair-pin loop-conformation of the probe.

In one embodiment, one, or a portion of both arms of the stem loop- structure hybridise to the target.

TEMBPERATURE

An-ϊmportani-aspect of the_present invention-is the- annealing temperature, and melting temperature of the probe.

Probe annealing temperature

As- used herein, the term "probe^anπealing temperature" refers to -the temperature at whieh the probe anneals to a target polynucleotide sequence under the experimental conditions preferred by the user.

Preferably, the term "probe annealing temperature" refers to the predicted temperature at which the probe anneals to a target polynucleotide sequence under the experimental conditions preferred by the user. The probe annealing temperature may be predicted using the various methods that are described herein. Typically, this temperature is calculated using computer analysis. The exact probe annealing temperature may be determined experimentally.

Preferably, the probe annealing temperature is at least 64⁰C

Preferably, the probe annealing temperature is not higher than 80⁰C.

Preferably, the.probe annealing temperature is not higher than 75°C.

-Preferably, the probe annealing temperature is in the range of from 64°C to 75°C, more preferably, from 64 °C to 74°C,_ more preferably, from- 64⁰C to 73⁰C,- more preferably, from 64⁰C to 72°C, more preferably, from 64⁰C to 71°C, more preferably, from 64⁰CtO 70⁰C, more preferably, from 64⁰C to ό^C_^more preferably, from 65⁰C to 74⁰C, more preferably, from 65°C to 73°C, more preferably, from 65°C to 72°C, more preferably, from 65⁰C to 71⁰C₅ more preferably, from 65°C to 7O⁰C, more j)referably,-frøm 65⁰C to 69⁰C, more preferably, from 66°C-to 74°C, more preferably, from 66°C to 73⁰C, more preferably, from 66°C to 72°C, more preferably, from 66°C to 71⁰C, more .preferably, from 6-6⁰G to 70⁰C, more preferably, from 66°C to 69°C, more preferably, from 67⁰C to 74°C, more preferably, from 67⁰C to 73⁰C, more preferably, from 67°C.to 72°C, more preferably, from ,67⁰C to-71°C, more preferably, from 67°C to.70-⁰C, more preferably, ^"from 67⁰C to 69-°C.

In one embodiment, the probe annealing temperature is 68 ⁰C.

In one embodiment,-the probe annealing temperatureSs 69 ⁰C-

In a further embodiment, the probe annealing temperature is 70- ⁰C.

These temperature determinations are based on the Beacon Design Software version 4 which uses the nearest neighbour thermodynamic calculations using Santa Lucia values (a unified view of polymer, dumbbell, and oligonucleotide DNA nearest- neighbour thermodynamics - Proc Natl Acad Sci U S A. 1998 Feb 17,95(4): 1460-5). Use of alternative means of calculating thermodynamic properties are available in the art.

Preferably, the melting temperature of the probe is less than the annealing temperature 5 of the probe.

Probe melting temperature

As used herein, the term "probe melting temperature" refers to the -temperature at -10 which -the closed- structure ofLthe probe - such as the hairpin-loop strueture of th& probe - is disrupted in 100%-of probes during an amplification reaction, preferably, a PCR reaction.

Preferably, the term- "probe melting temperature" refers to the predicted temperature

15 at which the closed structure- of the probe - such as -the hairpin-loop structure of the probe - is disrupted in.100% of probes during an amplification-reaction, preferably, a

PCR reaction. The probe melting temperature may be predicted using the various

-methods that .are described¹ herein. Typically, this temperature is calculated using computer analysis. The exact probe melting temperature may be determined

20 experimentally.

Preferably, the- probe melting-temperature .is-notiess than 45°C.

Preferably, J:he probe^meltihg temperatureJs- not-greater than 58°C

25

^■preferably.,Jlie probe melting temperature, is ^■ϊnthe_range of-from-45⁰-C-to-58-°C, more .preferably, from 45⁰C to 58⁰C, more preferably, from 46°C to.58°C, more preferably, from 47°C to 58°C, more preferably, from 48⁰C to 58°C, more preferably, from-49°C to 58°C, more preferably, from 50⁰C to 58°C, more preferably, from 51°C to 58°C,

30 more preferably, from 45⁰C to 57°C, more preferably, from 46⁰C to 57°C, more preferably, from 47°C to 57⁰C, more preferably, from 48°C to 57°C, more preferably, from 49°C to 57°C, more preferably, from 5O⁰C to 57°C, more preferably, from 51⁰C to 57°C, more preferably, from 45⁰C to 56°C, more preferably, from 46°C to 56°C, more preferably, from 47°C to 56°C, more preferably, from 48⁰C to 56°C, more preferably, from 49°C to 56°C, more preferably, from 5O⁰C to 56°C, more preferably, from 5FC to 56°C, more preferably, from 45⁰C to 55°C, more preferably, from 46°C to 55°C, more preferably, from 47°C to 55°C, more preferably, from 48°C to 55°C, more preferably, from 49-°C to 55⁰C, more preferably, from 50°C to 55⁰C, more preferably, from 51°C to 55°C, more preferably, from 45⁰C to 54°C, more preferably, from 46⁰C to 54⁰C, more preferably, from 47⁰C to 54°C, more preferably, from 48⁰C to 54⁰C, .more preferably, from 49°C to 54°C, more preferably, from 50⁰C to_54°C₅ more preferably, from 5FC to 54°C, more preferably_^ from 45°C to 53⁰C, more preferably, from 46°C to 53°C, more, preferably, from 47°C to 53°C, more preferably, from 48°C to 53^ΌC, more preferably,-from 49°C^~to 53⁰C, more preferably, from 50⁰C to 53°C, more preferably, from-5FC tΘ-53°Crmost preferably, 52 ⁰C

These temperature determinations are based on a linear DNA fold using the MFOLD server (http:/Λioweb.pasteur.fr/seqanal/interfaces/mfold.html) at 6ft ⁰C with a sodium concentration of 5OmM and Ά magnesium concentration of 3mM (Markham and _Zuker, 2005, NueK Acids. Res. 33 (web server issue)).

Use of alternative means of calculating thermodynamic^" properties of primers and probes are available in the_art - such- as Primer -3 (-see http://frodo.wi.mit.edu/cgi- ^" bin/pr-imer3/primer3_www.cgi) and ^"Visual OMP software (see" htfp://www.dnasθfh¥are..com/Pfodu^ htm.

In-one^" embodiment, the-nucleic acid probe has a rselting temgeratureJhat is greater man the annealing ^"temperature in ths--ampli^"fication reactionr Thus at the point at which -fluorescent emission -is monitored during an amplification reaetion,_any_ unbound probe will exist in the closed confirmation, having limited fluoresGεnt emission due to the proximity of the reporter molecule to-the quencher. ^"Furthermore, at the probe to template annealing temperature, the probe exists completely or almost completely in the open conformation and is therefore in the optimum conformation for hybridisation to the template.

Under these conditions the maximum amount of probe will be bound in an open conformation to the polynucleotide target sequence, as the reaction temperature is reduced, allowing the primers to hybridise. Also, the minimum background fluorescence is emitted at the amplification reaction. Hence, the present invention is both optimally hydrolysed and optimally quenched at different temperatures of the amplification -reaction.

5

In one embodiment the "read temperature" refers to the temperature at which the signal from the reporter-molecule is measured.

PRIMERS-

10-

"Suitably, the probe described herein-is used in conjunction with one or more primers. Suitably, the primer(s) are primers that can be used for the amplification of one or more nucleic acids.

15 In one embodiment, the primers have an annealing temperature of from about 52-58 °C. Preferably, the primer-s-lave an annealing temperature of from about 53-57 ⁰C, more preferably, the primers have an annealing temperature- of from about 54-56 ⁰C, most- preferably, the primers have an annealing temperature of about 55 ⁰C

20 Bpanother embodiment, the primers .have an annealing temperature of about 56.5 ± 1.5 ⁰C-

As used herein, the term "primer annealing temperature" refers to the temperature at which the primer anneals to a target polynucleotide sequence_under the experimental

-25 conditions preferred byihe user. Preferably, the term "primer anneaimg"temperature" refers to the predicted temperature at which the primer .anneals- to -a target polynucleotide sequence under the experimental conditions -preferred by the user. The primer annealing temperature may be predicted using the various methods that are described herein. Typically, this temperature is calculated using computer analysis.

30 The exact primer annealing temperature may be determined experimentally. Typically, the primers do not have a melting temperature (Tm) since they do not have any internal structure - such as a hairpin structure. Suitably, the primers are linear nucleic acid molecules without any internal structure.

In one embodiment, the primer parameters used are a length of 18 to 24 bp.

In one embodiment_^ the ampJicon length is -from 75 to- 150 bp.

In another embodiment, the target annealing temperature of the. probe is higher-than the target -annealing temperature of the primers. Without wishing to be bound^' by any particular theory, it is. believed that that as the temperature of the -amplification reaction ramps down the probe binds to the target nucleic before the primers bind. If the primers bind before the probe(s)^~then the polymerase used in- the amplification reaction may start to extend ^"from -the primer but the probe will not be in place to report what is happening in the amplification reaction.

REPORTER-QUENCHER PAIRS

Reporter-quencher pairs may be selected

xanthene dyes, Including fluoresceins, and rhodamine dyes. Many suitable forms of these compounds are widely available eommercially..

Preferably, theτeport is aϋluerescent reporter.

One group of fluorescent compounds-5J:e.theτiaphthylamines,^~having an amino group in the alpha-or beta position. Included among such-naphthylamino compounds- are 1- dimethylaminonaphthyl-5-sulfonate, l-anilino-8-naphthalene sulfonate and 2-p- touidinyl-6-naphthalene sulfonate.

Other dyes include S-phenyl-T-isocyanatocoumarin, acridities, such as 9- isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes, pyrenes, and the like. Reporter and quencher molecules may be selected from fluorescein and rhodamine dyes. These dyes and appropriate linking methodologies for attachment to oligonucleotides are described in for example, Histochemical J., 7: 299-303 (1975) and US 5,188,934.

Appropriate reporter-quencher pairs for particular probes as also described in Berhnan, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971)^ Griffiths,. Color and Constitution- of -Organic Molecules (Academic Press,- New York, 1976); Bishop, editor, Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbeok of Fluor-escent Probes and Research Chemicals (Molecular Probes, Eugene, 1992) Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949) and the like.

There are many linking moieties and methodologies for attaching-xeporter or qμencher molecules to the 5' or 3' termini of oligonucleotides, as described in Oligonucleotides and Analogues; A Practical Approach' (ERL Press, Oxford, 1-991); Nucleic Acids Research, 15: 5305-5321 (1987); Nucleic Acids Research, 19: 3019 (1991); PCR Methods and Applications, 2^223-227 (1993) and US -757, 141 and the like.

In one embodiment of the present invention,"the reporterJs located at the 5' end of the nucleic acid probe.

In one embodiment of the present invention,^~the reporter is located at the 3' end of the nucleic-acid profre.

In one embodiment of the present invention, the quencher is located at the 5^Lend_o£ the nucleic acid-probe.

In one embodiment of the present invention, the quencher is located at the 3' end of the nucleic acid probe.

In one embodiment of the present invention, the quencher is located internally within the nucleic acid probe. In one embodiment of the present invention, the reporter is located internally within the nucleic acid probe.

In one embodiment of the present invention, both the reporter -and "quencher -are located internally within the nucleic acid probe.

Preferably, the reporter is located at the 5' end of the nucleic acid probe -and the quencher is located at the 3' end of thejiucleic acid probe.

Preferably, the reporter is FAM.

Preferably, the quencher is Methyl Red.

DETECTION

In a further aspect of the present invention, there is provided-a method- for monitoring- or detecting a target nucleic acid in a sample comprising the use of a nucleic acid probe according-to-the present invention. The present invention is therefore suitable for monitoring_or detecting nucleic acids. -

The^~present invention is also suitable- "for monitoring or detecting- amplified^" nucleic acids. Accordingly, in^~a^~:iurtrϊer aspect, there-is provided_^a-method-for monitoring or detecting a target nucleic acid in an amplification reaction comprising the steps of: (a) contacting a sample of nucleic acid -with a nucleic_acid probe accordingJo the first aspect of the present invention;-(b);contacting said sample of nucleic acid- with one or more nucleic- acid- primers; (c) amplifying, the target nucleic acid; and (d) following and/or- during the amplification reaction measuring- the reporter activity.

Amplification methods include, but are not limited to PCR (as described in US 4,683,195; US4,683,202; and US4,965,188) and Real time PCR; the Ligase Chain Reaction (Genomics (1989) 4:560-569; Proc. Natl. Acad. Sci. U.S.A. (1991) 88:189- 193); the Polymerase Ligase Chain Reaction (PCR Methods and Applic. (1991) 1:5- 16); Gap-LCR (WO 90/01069); the Repair Chain Reaction (EP 439,182), 3SR (Proc. Natl. Acad. Sci. U.S.A. (1989) 86:1173-1177; Proc. Natl. Acad. Sci. U.S.A. (1990) 87:1874-1878; and WO 92/0880), and NASBA (US 5,130,238).

Suitably, the amplification reaction (eg. PCR or more preferably Real Time PCR) is performed at a denaturation temperature of about 95 ⁰C.

Suitably, the_amplifϊcation- reaction (eg. PCR or more preferably Real .Time PCR) is- performed at an extension temperature of about 72 ⁰C.

Suitably, the-amplifiυation reaction (eg. PCR ormore preferably Real Time PCR) -is_ performed^" at a read temperature- of from about 40-59 ⁰C, preferably, about 42-57 -⁰C, more preferably, about 44-55 °C, more preferably, about 46-53 ⁰C, more preferably, about 48-52 ⁰C, more preferably, about 49-51 ⁰C, most preferably, -about 50 ⁰C.

Suitably, thσ amplification reaction, (eg. PCR or more preferably Real Time PCR) is -performed at a read temperature of from about 45-59 ⁰C, preferably, about 45-57 ⁰G, more preferably,- about45-55 ⁰C, more preferably, about 45-53 ⁰C, more preferably, about 45-52^'°C, mote/preferably, about 45-51 ⁰C, more preferably, about 47-51 ⁰C.

In one embodiment, the amplification reaction (eg. -PCR or more--preferably Real Time PCR) is jperformed at a denaturation temperature of about 95 °C, a .read temperature o£about~50 ⁰C and-an extension-temperature^" of abou£32^~°C.

-In another embodiment, the-amplification reaction :(egJ3?-ΘR or jmore-preferably Real - ^"Time PCR) is performed at a- denaturation-temperature of-about-9.5 ⁰C^" for- about 15 seconds, an annealing temperature of about 50 ⁰C for about 30 seconds and an extension temperature of. about 72 ⁰C for ab.ouL30 seconds.

In another embodiment, the amplification reaction (eg. PCR or more preferably Real Time PCR) is performed using a hybridization step prior to the read temperature. Preferably, the temperature will be between about 55°C and about 80⁰C, preferably, between about 58°C and about 70⁰C. As described herein, a typical label is a fluorescent label. Fluorescence may be measured in a spectrofluorometer - such as a Hitachi/Perkin Elmer Model 650-40 (Perkin Elmer, Norwalk, Conn.) or a PTI LS-100 Luminescence Spectrophotometer (Photon Technology International, London, Ontario, Canada). A spectrofluorometer, depending on the features of the particular machine utilised, offers the opportunity to set the excitation and emission wavelength, as well as bandwidth. Although each label has a discrete fluorescence spectrum, a broad range of detection wavelengths are suitable for practising the present invention.

Fluorescent measurements may be carried-out before and after the reaction has been performed. The change in fluorescence may be calculated relative- to the pre-reaction value. Equivalently, a portion of the reaction mixture may not be subject to the reaction-conditions. In this manner, the pre-reaction fluorescence- can be- measured, together with the post-reaction fluorescence, -after completion of the reaction. The- use of- reaction vessels- which are also suitable for use in measuring fluorescence allows diierct measurements of both pre-and post-reaction fluorescence without opening the -reaction vessel or other post-reaction manipulations.

In -some methods, tike amplification- reaction may be carried out as. an automated process. Thermal cyclers- are currently available ^"from, for example, Perkin Elmer (Norwalk, Gorm.) that uses a heat block- capable of holding up to 48,-96- or 384 reaction tubes. Consequently, up to 3-84-amplification -reactions can be carried Oufc- simultaneously.

Advantageously, the -present invention enables the -automatic detection of FCR products in all samples_* without the need to handlerthe-samples, open-the-tuhes_r or^~ ϊnterrupt the cycling reaction, using for example, suitable optical systems. In one such optical system, multiple fibre optic leads are used to transmitihe excitation light from the source to the reaction tube and measures the emission light from each tube. Only a single fluorometer is needed to read fluorescence from the reaction tubes, as each fibre optic can be read rapidly one at a time. An alternative optical system, a video camera may be used to measure the fluorescence of multiple reaction vessels simultaneously. The use of transparent reaction vessel tops allows the measurement of fluorescence without opening the vessel. An alternative suitable detection scheme comprises the use of a 96 or 384-well microtiter format. This type of format is frequently desirable in clinical laboratories for large scale sample screening, for example, for genetic analysis.

The methods of the present invention can be used to simultaneously detect multiple target sequences. Probes specific to each target are- present in the reaction mixture. Eor each target nucleic acid present in the sample, the corresponding probe will hybridise and he cleaved. In order to detect the cleaved probes separately, each species of probe may be labeled -with-a. label that fluoresces at a distinct wavelength. Each species of probe_may then be detected separately by suitable selections of the measured wavelength.

Thus, the. methods of the present invention are also useful for detecting the amplification products in co-amplification methods for detecting several targets in one. sample without ever opening the reaction vessel once the amplification reaction-is initiated. The present invention can even Jbe usedςfor quantitative comparisons of two different nucleic acid targets in the same sample. Methods for quaπtitating nucleic acids are described in_^for example, US 5_^219,727.

SAMPDE

In general, the nucleic acid in the sample wiil^~be a sequence of DNA, mostiypically genomic DNA.- Suitable nucleic acid-samples also include single or double-stranded DNA or RNA^~ The present invention can-also be^used-with Othernucleic acids - such - as-messengerRNA,-ribosomal RNA, viral -RNA, or-cloned DNA.

Sample preparation wilhvary depending on the source of the sample, the target to be detected, and the reaction used. Suitable sample preparation protocols are known in the art and described in the literature cited herein (e.g., Sambrook et al.). Simple and rapid methods of preparing samples for the PCR amplification of target sequences are described in Higuchi (1989) in PCR Technology (Erlich ed., Stockton Press, New York), and in PCR Protocols, Chapters 18-20 (Innis et al., ed., Academic Press, 1990). HYBRIDISATION

As used herein, the term "hybridisation" refers the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing.

Hybridisation can -occur between fully complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Conditions -under which only fully complementary nucleic acid strands will hybridise are referred to as "stringent- hybridisation- conditions". Two single-stranded nucleic acids that are complementary except for minor regions of mismatch are referred to as-^substantially complementary". Stable duplexes of substantially complementary sequences can be achieved under less stringent

conditions. Those skilled in the art of nucleic -acid technology can determine duplex stability empirically considering a number of variables including, for-example,-the length and base paireoncentration of the oligonucleotides, ionic strength, and-incidence of mismatched base pairs.

NUCLEIC ACID

Aspects αfthe-present invention involve the use of nucleotide sequences, which maybe-available in databases.

The nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin. The nucleotide sequence "may be double-stranded or single- stranded whether representing The sense or antisense-strand-or combinations thersofi

The nueleotide sequence may be DNA.

The nucleotide sequence may be prepared by use of recombinant DNA techniques (e.g. recombinant DNA).

The nucleotide sequence may be cDNA. The nucleotide sequences may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or pσiylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art.

KITS

The materials for use in the methods of the present invention are ideally suited -for preparation ofKits.

In one aspect, there is provided- a kit comprising one or more nucleic acid probes as described herein and^~a nucleic acid polymerase.

Such a kit may further comprise one or more of the various reagents (typically in concentrated form) utilised in the methods, including, for example, ^"buffers and the appropriate nucleotide triphosphates -(erg., dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP).

The components of the-kit of-the present invention may be present itLcontainer-s.

Θligonucleotides-can be^~in-any form, -e.g., lyophilized, or in-solution ^"(e.g.,- a distilled⁷ water or buffered solution), etc.-Oligonucleαtides ready for use in, for example, the same -amplification reaction may be combined -in_.a single- container-υr can be -in separate containers.

The kit optionally further comprises one or more controls - such as one or more control nucleic acids.

The kit optionally further comprises one or more primers as described herein.

A set of instructions will also typically be included. GENERAL RECOMBINANT DNA TECHNIQUES

The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant -DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch,. and T. Maniatis, 1989, Molecular Cloning: A Laboratory- Manual, Second- Edition, Books 1-3, .Cold Spring Harbor Laboratory Press; Ausubel, F. M. et-al. (1995 and periodic supplements; Current Protocols. in-M&lecular Biology, ch. 9, 1.3, and -16, John Wiley & -Sons, New- York, N. Y.); B. Roe, J.=Crabtree;_and A. Kahn, 199β_s DNA Isolation and Sequencing: Essential Techniques; John Wiley & Sons]" M. -J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IrI Press; and, D. M. ff. Lilley and J. E. Dahlherg, 1992, Methods- of Emymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic. Press. Each of these general texts is herein incorporated by reference.

The Invention will now be further described by way of Example, which are meant to serve to- assist one of ordinary skill in the art in carrying out the invention and are not intended in anyway to lirnit-the scope of the invention.

EXAMPLES

EXAMPLE l

Materials and-Meifiods^÷ Primer -Design

Primer and probes "for Taqman and Molecular BeaGon-assays were designed using Beacon Designer (Premierbiosoft). Taqman probe with 3' and or 5' additions (TaqTag probes) maintained the exact same probe template annealing structure to minimise such that apart from the sequence independent tags, the probes were identical. Four of the Taqman/TaqTag comparison probes maintained identical 5' terminal sequences and for those that had slightly different 5' ends, terminal G residues were avoided as these are know to exert a quenching effect. Molecular Beacon probes were shorter than the Taqman probes but the probe sequence was designed within the footprint of the taqman probe. Taqman sequences and were modified using two strategies. Firstly, Bases complimentary to the 5 'end were added to the 3 'end to form a stem loop structure. In a second system, a sequence^"

5 independent stem loop was created by adding Gs and Cs to the 5' and V ends of the probe analogous to the Beacon stem loops. The melting a points of these probes was predicted using the. mFOLD server

(http:^ioweb.pasteur.fr/seqanal/interfaces/mfold,htmπ with the salt- concentrations set at, Na+ = 5OmM, Mg++ = 3mM.

10.

OligQ-Svnthesis

The-oligonucleotides for this study were sythesised-by ADTbio (SouthamptoniJK). Double labelled reporter probes were labelled with a 5'FAM and-3'dabcyl-quencher. 15

Melt Curves

Melt curves were run before-and after Real-Time PCR amplification to establish the conformation and structure and melting points of the. different probes. Melt curves 20 —were rurron the icyclerlQ-between 35°C and 95°C with_a-ranrp rate of 0.5⁰C every 10 seconds and_a reading interval ofO.5°C

-Real-Time PCR

25 Real-Time PGR amplification plots were acquired-on ihe-Tcycler IQ -system. (Bio- R-ad). Except where noted, the cycling- conditions were 95°G for 8mins followed by 62 cycles-of 95⁰C for 15 seconds, 5€°C for 30s and 72^ΌC fori5s. Fluorescence was read during the 5O⁰C incubation. Where different hardware platforms were used, the- ramping rates differed between the machines, icyclerlQ 3 ⁰C / second, Lightcycler

30 10⁰C / second and the rotorogene on the default setting which gives rapid but non linear ramping.

Each PCR reaction contained 50OnM of forward and reverse primer and 20OnM of probe in a 15ul reaction that also contained 7.5ul or 2X PCR core mix (Eurogentec, Belgium) and 25ng of human rhabdomyosarcoma cell cDNA, 12.5ng of human genomic DNA, or 25ng of HeLa cell cDNA 8 hours post infection with human RV14 at an M.O.I of 2. DNA and RNA was purified on qiagen spin column purified using standard -protocols (Qϊagen).

Data processing

icyclerlQ

Baseline cycles were set automatically for each well. The threshold was set automatically- at 1OX the_ standard deviation from the background level. ^• This collected data from the exponential^phase of. all traces.

^'Rotorgene

Data acquired from the rotegene using the recommended setting for normalising the data T.e dynamic tube and slope correction both activated. The threshold was set manually early in the exp@nential^~phase-on the logarithmic plot.

Lighteycler

Data was normalised- using" the arithmetic method for baseline correct and -CT values were obtained usϊngjhe fit points protocol accerdingto the manufactures protocols.

EXAMPLE 2

Improvement of-the performance of a_probe-to detect the asthma-associated alternative splise-varfant 1 target sequence (AAAl). The protocols above were-used to-^~design and-test probes of different parameters.

The Probes used in this-study are shown in Table 1.

The results are presented in Figures 1-6 and the legends thereto.

EXAMPLE 3 The probes of the present invention are compared to the performance of Taqman and Molecular Beacon probes for a range of target sequences and using a variety of different real-time PCR platforms.

The probes used for this study are shown in Table 1.

The results are presented in Eigures 7 and 8 and the legends thereto.

The data show that the design features described herein, result in- a probe conformation that is more sensitive at detection amplification than Taqman and^" Molecular Beacon probes.

FURTHER ASPECTS

The -present invention relates to-the use of the oligonucleotide probe described- herein for the monitor-ing of nucleic acid amplificatioru

The present invention relates to- the monitoring of^~nucleic acid amplification of a target sequence using a_nucleic acid polymerase ^"Saving 5 '-3' nuclease activity and a- primer capable of hybridising to the target sequence, and the present invention being an oligonucleotide probe-capable of hybridising-with the target sequence 3' relative to- the primer.

The present invention provides a process for- replication- and homogeneous -assay detection of a "target nucleic acicL-sequence in .a^~sample, said process comprising ther _^steps of:

a) a PCR reaction mixture, a sample containing or suspected to contain a- target nucleic acid sequence, oligonucleotide PCR primers that hybridize to opposite strands of the target nucleic acid sequence and flank the target sequence for PCR amplification of said target sequence, each of four deoxynucleoside triphosphate bases and nucleic acid polymerase having 5' to 3' exonuclease activity and devoid of 3' to 5' exonuclease activity. b) amplifying the target polynucleotide sequence in the sample under suitable PCR reaction mixture temperature conditions by a repetitive series of PCR thermal cycling steps comprising:

1) denaturing the target nucleic acid sequence into opposite strands;

2). hybridising the oligonucleotide probe within the target nucleic acid sequence of the -denatured- strands and hybridising the, oligonucleotide PCR primers to the denatured strands, and

3} extending the hybridised primers-with me four deoxynucleoside triphosphate bases and the nucleic acid polymerase, and producing reporter molecule labelled nucleotide fragments during this extension _.phase -by the- 5' to 3' exonuclease activity of -the nucleic acid polymerase on probe annealed to the denatured strands;

a) following and or during amplification of-the target nucleic acid sequence by one or more series of thermal cycling" recording-the amount of fluorescent emission by the reporter molecule as a -measure of the rate of amplification of the target polynucleotide sequence.. Additionally the -fluorescent emission from_-the ^reporter molecule can be-measured relative to "the emission from the quencher molecule.by ^"fluorescent polarisation.

Table 1

This lable shows the Taqman and Molecular Beacon probes used^" in Figures- 1 to 8. Sequences in Black represent the Probe template annealing, sequences. Some of the probes have 5' additional bases. Some of the probes -have 3' additional -bases. Some of thejsrobes have both 5' and 3' additional basesr

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications- of the- described modes, for carrying out the invention which are obvious to those skilled in molecular biology or -related fields are-intended to be withui-the scope of the fellowing_claims.

Claims

1. A nucleic acid probe for monitoring or detecting a target nucleic acid in a sample, wherein said probe:

(i) comprises a reporter molecule and a quencher molecule capable of quenching the reporter molecule;

(ii) has a substantially closed- structure inits unhybridised state such ihat the reporter is substantially quenched;

(iii) is modified at the 3' terminus to prevent extension by a nucleic acid polymerase; and

(iy) is or is capable of being substantially hydrolysed by the nucleic acid polymerase,

and wherein the nucleic acid probe has:

(a) a target annealing temperature of-ffom about 64 ⁰C to about 75 °C; and

(b) a melting temperature of fromabout 45_°C^"to about 58 ⁰C.

2. The nucleic acid probe according to claim 1, wherein the closed structure is a hairpin-loOp structure.

3. The-nucϊeic probe according to elaim=l or claim 2, wherein the- proximity of the quencher to the reporter is reduced when the probe is at -the target -annealing temperature.

4. The nucleic acid probe according to any of the preceding claims, wherein the reporter is a fluorescent reporter.

5. The nucleic acid probe according to any of the preceding claims, wherein the quencher is a fluorescent quencher.

6. The nucleic acid probe according to any of the preceding claims, where said probe is or is capable of being substantially hydrolysed by a nucleic acid polymerase having

5' to 3' exonuclease activity.

7. The nucleic acid probe according to any of the preceding claims, wherein the-probe is. completely-hydrolysed:

8. The nucleicaeid probe according to- any of the preceding claims, wherein the probe comprises one or more additional nucleic hases at the 5' end of the oligonucleotide.

9. The nucleic acid probe according to any of the preceding claims, wherein the .probe comprises one or more- -additional nucleic bases at the 3' end of the oligonucleotide.

IGF. The nucleic acid probe according, to any of the preceding claims, wherein the probe, comprises one^" or mere- additional nucleic bases .at the.5' and 3' ends- of the - oligonucleotide that are_compTementary-to-each othen

11. A method for monitoring- or detecting a target nucleic acid in a sample" Gomprisingithe use-of a nucleicaeid probe, according to -any of-claims MO.

12. A method forrmonitoring or detecting a-target nucleic acidJn an amplification reaction comprising the steps-ef:

(a) contacting a sample of nucleic acid with a nucleic acid probe according to any of claims 1-10 and one or more nucleic acid primers;

(b) amplifying the target nucleic acid; and

(c) following and/or during the amplification reaction measuring the reporter activity.

13. The method according to claim 12, wherein the annealing temperature of the probe is higher than the annealing temperature of the primer(s).

14. The method according to claim 12 or claim 13, wherein the annealing temperature of the primers is from about 52-58 ⁰C.

15. The method according to claim 12-or -claim 13, wherein the read temperature of -the amplification reaction is from 40-59 ⁰C.

16. The-method according to any of claims 12_to 15, "wherein- said .probe hybridises to the target sequence 3' relative to the one or more primers.

17. The method according to any of claims 12 to 16, wherein the primers are linear DNA molecules with no internal structure.

18. The- method according to any of claims 12 to 17, wherein the nucleic acid is amplifϊednsing PCR.

19. The method according to any of claims 12-1S₅ wherein- the nucleic acid is

20. The method according to any of claims 12-19, wherein the-amplification reaction is-performed at a denaturaticn- temperature of .about 95^~°C7 an annealing temperature of about 50 "Oandarread temperature of about 72 ⁰C.

21. The method according to any of claims 12-20, wherein the reporter activity is measured during the annealing stage of the amplification reaction.

22. A method for preparing a nucleic acid probe for monitoring or detecting a target nucleic acid in a sample, wherein said probe: (i) comprises a reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule;

-(ii) has a substantially closed structure in its unhybridised state such that the reporter is substantially quenched;

(iv) is or is capable of being substantially hydrolysed by the nucleiαacid polymerase,

comprising the steps of:

(a) identifying a nucleic acid- probe sequence that can be used to monitor or detect a -target nucleic acid;

-(b) adjusting-the design-of the nucleic acid "pr-obe- such that the annealing temperature of said probe is from about 64 ⁰C to about 75 °C; and

(c) adjusting the design of the-nucleic acid_probe-such that the melting-temperatute of said probe is from abuut45 ⁰C to about 58⁰G.

23. Use of the nucleic acid probe according to any of claims 1-10 for monitoring or -detecting-a target nucleic-acid irra samplβ.

24. A ^"Kit comprising a nucleic acid probe. aeGer-ding to any of claims 1-1-0, a-nucleic acid polymerase and optionally, one or more primers.