Europäisches Patentamt *EP000975805B1* (19) European Patent Office Office européen des brevets (11) EP 0 975 805 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.7: C12Q 1/68 of the grant of the patent: 05.09.2001 Bulletin 2001/36 (86) International application number: PCT/GB98/01057 (21) Application number: 98917331.5 (87) International publication number: (22) Date of filing: 09.04.1998 WO 98/46790 (22.10.1998 Gazette 1998/42) (54) HYBRIDISATION ASSAY IN WHICH EXCESS PROBE IS DESTROYED HYBRIDISIERUNGSVERFAHREN IN DEM ÜBERSCHÜSSIGE SONDEN ABGEBAUT WERDEN TEST D’HYBRIDATION DANS LEQUEL LA SONDE EN EXCES EST DETRUITE (84) Designated Contracting States: (56) References cited: AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU EP-A- 0 144 913 EP-A- 0 780 479 MC NL PT SE WO-A-90/01559 WO-A-98/19168 US-A- 4 833 084 (30) Priority: 14.04.1997 GB 9707531 • DATABASE WPI Derwent Publications Ltd., (43) Date of publication of application: London, GB; AN 92205018 XP002072949 "gene 02.02.2000 Bulletin 2000/05 detection method-comprises gene extn., denaturing to single strand, specifically bonding (73) Proprietor: Zetatronics Limited to labelled gene probe and reacting" & JP 04 135 Hatfield, Herts AL10 9AB (GB) 498 A (TOSHIBA) , 8 May 1992 • HARBRON S. ET AL.,: "Amplified assay of (72) Inventor: Harbron, Stuart alkaline phosphate using Flavinadenine Berkhamsted, Herts HP4 2LL (GB) dinucleotide phosphate as substrate" ANALYTICAL BIOCHEMISTRY, vol. 206, - 1 (74) Representative: Coates, Ian Harold et al October 1992 pages 119-124, XP002072948 Sommerville & Rushton, 45 Grosvenor Road St. Albans, Hertfordshire AL1 3AW (GB) Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 975 805 B1 Printed by Jouve, 75001 PARIS (FR) 1 EP 0 975 805 B1 2 Description ence of the label in the resulting hybrid is determined. A disadvantage of this method is that it is neither easy Background Art nor convenient to attach the single-stranded target nu- cleic acid to a solid support, the whole process involving [0001] Nucleic acid hybridisation is a widely used 5 a 12 - 15 hour incubation of the nucleic acid with a nitro- technique for identifying, detecting and quantitating tar- cellulose sheet, followed by a 2 hour baking step. This get polynucleotide sequences in a sample. This tech- makes the assay slow and unattractive for routine use. nique relies for its success on complementary base pair- It is also cumbersome, with the hybridisation and wash- ing between the two halves of a double-stranded nucleic ing steps being carried out in a sealed pouch, containing acid molecule: when single-stranded nucleic acids are 10 the membrane and the buffer solution. In addition, when incubated in solution under suitable conditions of tem- very low concentrations must be detected, the ratio of perature, pH and ionic strength, complementary base specific to non-specifically bound probe can be very low sequences pair to form double-stranded stable hybrid and repeated washing under highly stringent conditions molecules. This ability of single-stranded nucleic acid is frequently required. Under these conditions the sen- molecules to form a hydrogen-bonded structure with 15 sitivity of the assay is often compromised because of their complementary nucleic acid sequences has long substantial loss of specifically bound probe. been employed as an analytical tool in recombinant [0005] Since then a many improvements have been DNA research. made, most of which employ a sandwich approach us- [0002] In most cases the sample will contain double- ing two probes: a reporter probe and a capture probe. stranded nucleic acid and must be denatured prior to 20 The reporter probe is a nucleic acid having a sequence the hybridisation assay to render it single-stranded. A complementary to at least part of the target sequence nucleic acid having a known sequence which is comple- and which is labelled with a detectable group. The cap- mentary to the target sequence is either synthesised ture probe is a nucleic acid having a sequence comple- chemically in an automated fashion with great facility, or mentary to at least part of the target sequence, but which is isolated from the appropriate organism and rendered 25 is different to that of the reporter probe, and which is single-stranded by denaturation. It is then used as a labelled with an immobilisable group. In many applica- probe to search a sample for a target complementary tions, pairs of specific binding members (sbm's) have sequence. Detection of specific target nucleic acids en- been used for this purpose. ables accurate diagnosis of bacterial, fungal and viral [0006] For example, in U. S. Pat. No. 5,273,382 to disease states in humans, animals and plants. Addition- 30 Snitman and Stroupe a capture probe complementary ally, the ability to probe for a specific nucleotide se- to part of the target nucleic acid is labelled with an anti- quence enables the diagnosis of human genetic disor- gen or antibody. After hybridisation between this capture ders. Hybridisation produces stable hybrids, and a probe and the target, the solution is introduced to a sup- number of different approaches are known to the art for port-bound antibody or antigen which immobilises the detecting these. 35 hybrid formed between the capture probe and the target. [0003] One approach involves the use of labelled Following a washing step, a second, reporter probe, probes. By labelling a probe nucleic acid with some complementary to a different region of the target nucleic readily detectable chemical group, it is possible tc detect acid, is introduced and the triple sandwich formed is de- the polynucleotide sequence of interest in a test medium tected. containing sample nucleic acids in single-stranded form. 40 [0007] Similar approaches are described by Holtke et Nucleic acids have been labelled with radioisotopes, en- al.: in U.S. Pat. No. 5,344,757 is disclosed a method in zymes and fluorescent molecules. The use of labelled which a reporter probe is labelled with digoxin or digox- nucleic acids as probes in macromolecular analysis is ygenin, and hybrids are captured using antibodies important for clinical, veterinary and environmental di- against this hapten. In this case, a capture probe is not agnostic applications. 45 used, and the method is limited either to the detection [0004] Early methods for detecting target nucleic ac- of an immobilised target, or when the assay is used for ids involved their immobilisation on a solid support such detecting PCR products, one of the primers is immobi- as nitro-cellulose paper, cellulose paper, diazotized pa- lised. In U.S. patent 5,354,657 the method is further de- per, or a nylon membrane. For example, in U. S. Pat. veloped and involves the solution hybridisation between No. 4,358,535 to Falkow a method is disclosed in which 50 the target nucleic acid and a reporter probe labelled with the target nucleic acid is rendered single-stranded and digoxin or digoxygenin. This hybrid is captured by a sol- then immobilised onto a membrane. A labelled probe id-supported capture probe, complementary to a differ- which is complementary to the target nucleic acid is ent region of the target. A detectably labelled antibody brought into contact with the solid support and hybridis- against the hapten is then added and the hybrids formed es to the target nucleic acid. The solid support is washed 55 detected. several times at a carefully controlled temperature to re- [0008] Specific binding members other than antigens move unbound and non-specifically bound probe with- or haptens and antibodies have been used. In U.S. Pat. out removing specifically bound probe, and the pres- No. 5,374,524 to Miller is described a method for the 2 3 EP 0 975 805 B1 4 solution sandwich hybridisation, capture and detection reagent is selective for DNA-RNA or RNA-RNA hybrids of amplified nucieic acids. Amplicons are denatured and over the single-stranded nucleic acids. U.S. Pat. No. treated with an enzyme-labelled reporter probe and a 5,200,313 to Carrico further discloses a nucleic acid hy- biotinylated capture probe. Hybrids formed are captured bridisation assay employing an immobilised or immobi- using streptavidin-coated chromium dioxide particles. 5 lisable polynucleotide probe selected to form DNA-RNA [0009] Disadvantages of these approaches include or RNA-DNA hybrids with the particular polynucleotide the increased cost and complexity of using two probes. sequence to be determined. Resulting hybrids are de- For example, for each assay two probes need to be syn- tected by binding of an antibody reagent, preferably la- thesised and labelled: one for use as the capture probe, belled with a detectable chemical group, selective for and the other for use as a reporter probe. In addition, 10 binding the hybrids in the presence of the single-strand- hybridisation conditions have to be carefully chosen to ed sample and probe nucleic acids. Advantageous fea- form the sandwich of target, capture probe and reporter ture of Carrico's inventions are that no immobilisation or probe. labelling of sample nucleic acids is required and hybrid- [0010] Simpler approaches which avoid the use or a isation can be performed entirely in solution. A further capture probe have been described. Atlas and Steffan 15 advantage is that a universal detection reagent may be (Biotechniques (1990) 8:316 - 318) disclose a solution used whatever the target is.