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Hybridization Probes for Nucleic Acid Detection, Universal Stems, Methods and Kits

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Hybridization Probes for Nucleic Acid Detection, Universal Stems, Methods and Kits (19) & (11) EP 2 423 322 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 29.02.2012 Bulletin 2012/09 C12Q 1/68 (2006.01) C07H 21/00 (2006.01) (21) Application number: 10184995.8 (22) Date of filing: 14.11.1994 (84) Designated Contracting States: • Tyagi, Sanjay AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL New York, NY 10038 (US) PT SE • Lizardi, Paul Wallingford, CT 06492 (US) (30) Priority: 12.11.1993 US 152006 (74) Representative: Wilkinson, Marc George et al (62) Document number(s) of the earlier application(s) in avidity IP accordance with Art. 76 EPC: Merlin House 07075511.1 / 1 921 169 Falconry Court 95904104.7 / 0 728 218 Baker’s Lane Epping, Essex CM16 5DQ (GB) (71) Applicant: PHRI Properties, Inc. Newark, NJ 07107-3001 (US) Remarks: •Claims filed after the date of receipt of the divisional (72) Inventors: application (Rule 68(4) EPC). • Kramer, Fred Russell •This application was filed on 30-09-2010 as a Riverdale, NY 10463 (US) divisional application to the application mentioned under INID code 62. (54) Hybridization probes for nucleic acid detection, universal stems, methods and kits (57) Unitary hybridization probes for the detection of conformation. The shift is detectable due to reduced in- nucleic acid target sequences comprise a target comple- teraction of the label pair. The unitary probes may be ment sequence, an affinity pair holding the probe in a unimolecular or bimolecular. Also, universal stems and closed conformation in the absence of target sequence, kits useful for constructing said probes. Also, assays uti- and a label pair that interacts when the probe is in the lizing said probes and kits for performing such assays. closed conformation. Hybridization of the target and tar- get complement sequences shifts the probe to an open EP 2 423 322 A1 Printed by Jouve, 75001 PARIS (FR) 1 EP 2 423 322 A1 2 Description ground signal. Monitoring requires sophisticated instru- ments, and, even so, sensitivity is limited. Moreover, the [0001] The present invention relates to the field of as- hybridization signal is, in some cases, a negative one; says which involve nucleic acid hybridization probes. As- i.e., the presence of target results in a reduction in the says are useful for the detection of specific genes, gene 5 amount of fluorescence measured at a particular wave- segments, RNA molecules and other nucleic acids. Such length. assays are used clinically, for e.g., tissue, blood and urine [0005] This technique requires that two unassociated samples, as well as in food technology, agriculture and probes bind simultaneously to a single-stranded target biological research. sequence. The kinetics of this tri- molecular hybridization 10 are too slow for this technique to be suitable for real- time Background of the Invention detection. The requirementthat target be single- stranded makes the technique unsuitable for in vivo detection of [0002] Nucleic acid hybridization probes are used to double-stranded nucleic acids. detect specific target sequences in a complex mixture. [0006] Another solution-phase scheme also utilizes a Conventional, heterogeneous, hybridization assays,15 pair of oligodeoxynucleotide probes. However, here the such as those described in Gillespie and Spiegelman two probes are completely complementary both to each (1965), typically comprise the following steps: immobili- other and to complementary strands of a target DNA zation of at least the target nucleic acid on paper, beads, (Morrison, 1987; Morrison, 1989; Morrison et al., 1989; or plastic surfaces, with or without using capture probes; Morrison and stols, 1993). Each probe includes a fluor- addition of an excess of labelled probes that are comple- 20 ophore conjugated to its 3’ end and a quenching moiety mentary to the sequence of the target; hybridization; re- conjugated to its 5’ end. moval of unhybridized probes; and detection of the [0007] When the two oligonucleotide probes are an- probes remaining bound to the immobilized target. nealed to each other, the fluorophore of each probe is [0003] Unhybridized probes are removed by extensive held in close proximity to the quenching moiety of the washing of the hybrids. This is generally the most time- 25 other probe. If the fluorescent label is then stimulated by consuming part of the procedure, and often utilizes com- an appropriate frequency of light, the fluorescence is plex formats such as sandwich hybridization. The use of quenched by the quenching moiety. However, when ei- solid surfaces lengthens the time it takes for hybridization ther probe is bound to a target, the quenching effect of by restricting the mobility of, or access to, the target. The the complementary probe is absent. The probes are suf- largearea presented by the solid surfaces nonspecifically 30 ficiently long that they do not self-quench when target- retains unhybridized probes, leading to background sig- bound. nal. Additionally, solid surfaces may interfere with signal [0008] In this type of assay, there are two opposing from the probes. The requirement that the probe-target design considerations. It is desirable to have a high con- hybrids be isolated precludes in vivo detection and con- centration of probes toassure that hybridization of probes current detection of nucleic acids during synthesis reac- 35 to target is rapid. It is also desirable to have a low con- tions (real-time detection). centration of probes so that the signal from probes bound [0004] Several solution-phase detection schemes, to target is not overwhelmed by background signal from sometimes referred to as homogeneous assays, are probes not hybridized either to target or other probes. known. By "homogeneous" we mean assays that are per- This situation necessitates waiting a relatively long time formed without separating unhybridized probes from40 for the background fluorescence to subside before read- probe-target hybrids. These schemes often utilize the ing the fluorescent signal. fact that the fluorescence of many fluorescent labels can [0009] An assay according to this scheme begins by be affected by the immediate chemical environment. One melting a mixture of probes and sample that may contain such scheme is described by Heller et al. (1983) and also targetsequences. The temperature is then lowered, lead- by Cardullo et al. (1988). It uses a pair of oligodeoxynu- 45 ing to competitive hybridization. Some probes will hybrid- cleotide probes complementary to contiguous regions of ize to target, if present; some will hybridize to comple- a target DNA strand. One probe contains a fluorescent mentary probes; and some will not hybridize and create label on its 5’ end and the other probe contains a different background signal. A parallel control assay is run with fluorescent label on its 3’ end. When the probes are hy- no target, giving only a background level of fluorescence. bridized to the target sequence, the two labels are very 50 If the sample contains sufficient target, a detectably high- close to each other. When the sample is stimulated by er level of residual fluorescence is obtained. light of an appropriate frequency, fluorescence reso- [0010] With this scheme it is necessary to delay read- nance energy transfer ("FRET") from one label to the ing the residual fluorescence for a considerable time to other occurs. This energy transfer produces a measura- permit nearly all the excess probes to anneal to their com- ble change in spectral response, indirectly signaling the 55 plements. Also, a parallel control reaction must be per- presence of target. The labels are sometimes referred to formed. Additionally, a low concentration of probes is as FRET pairs. However, the altered spectral properties used to reduce the fluorescent background. Thus, kinet- are subtle, and the changes are small relative to back- ics are poor and result in an inherently slow assay. That 2 3 EP 2 423 322 A1 4 precludes real-time detection. These problems are par- be used to make such nucleic acid probes specific for ticularly severe for double- stranded targets. The probes, target sequences of choice. as well as the targets, need to be melted, rendering the [0017] A further object of this invention is homogene- assay unsuitable for use in vivo. Also, the signal is not ous assays using such probes. only residual, it is a differential signal from comparison 5 [0018] A further object of this invention is hybridization to an external control. probes and rapid methods wherein detection is per- [0011] Another solution-phase scheme utilizing the formed quickly and without delay. phenomenon known as strand displacement is described [0019] A further object of this invention is hybridization by Diamond et al., 1988. Typically, these assays involve probes and methods that can detect nucleic acids in vivo. a bimolecular nucleic acid probe complex. A shorter sin- 10 [0020] A further object of this invention is hybridization gle-strand comprising a subset of the target sequence is probes and methods that can detect nucleic acids in situ. annealed to a longer probe single- strand which compris- [0021] A further object of this invention is hybridization es the entire target binding region of the probe. The probe probes and methods that can detect nucleic acid target complex reported thus comprises both single-stranded sequences in nucleic acid amplification and other syn- and double-stranded portions. The reference proposed 15 thesis reactions in real-time mode. that these probe complexes may further comprise either [0022] A further object of this invention is hybridization a 32P label attached to the shorter strand or a fluorophore probes and assays that permit detection of nucleic acid and a quencher moiety which could be held in proximity targets without the use of expensive equipment. to each other when the probe complex is formed. [0023] Inorder to realize the full potentialof the process [0012] It is stated that in assays utilizing these probe 20 of hybridization in the field of diagnostics and research, complexes, target detection is accomplished by a two- a technique is needed for monitoring hybridization in so- step process.
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