USOO6110678A United States Patent (19) 11 Patent Number: 6,110,678 Weisburg et al. (45) Date of Patent: Aug. 29, 2000 54 TWO-STEPHYBRDIZATION AND OTHER PUBLICATIONS CAPTURE OF A POLYNUCLEOTIDE Arnold et al., “ASSay Formats Involving (75) Inventors: William G. Weisburg; Jay H. Shaw; Arcidinium-Ester-Labeled DNA Probes’, Clinical Chemis Michael M. Becker; Mehrdad Majlessi, all of San Diego, Calif. try, 35(8):1588–1594 (1989). Iwen et al., “Evaluation of Nucleic Acid-Based Test (PACE 73) Assignee: Gen-Probe Incorporated, San Diego, 2C) for Simultaneous Detection of Chlamydia trachomatis Calif. and Neisseria gonorrhoeae", J. Clin. Microbiology, 33(10):2587-2591 (1995). Appl. No.: 09/070,998 Lund et al., “Assessment of Methods for Covalent Binding Filed: May 1, 1998 of Nucleic Acids to Magnetic Beads, Dynabeads.TM, and the Characteristics of the Bound Nucleic Acids in Hybridization Related U.S. Application Data Reactions”, nucleic Acids Research, 16(22):10861-10880 60 Provisional application No. 60/045,430, May 2, 1997. (1988). 51 Int. Cl." ........................... C12Q 1/68; CO7H 21/02; C07H 21/04; C12N 15/00 Moore et al., “Amplification of rRNA for Assessment of 52) U.S. Cl. ............................ 435/6; 536/23.1; 536/24.3; Treatment Response of Pulmonary Tuberculosis Patients 935/76; 935/77; 935/78 during Antimicrobial Therapy”, J. Clin. Microbiology, 58 Field of Search ..................... 435/6, 91.2, 536/23.1, 34(7): 1745–1749 (1996). 536/24.3; 935/76, 77, 78 Nelson et al., “Detection of all Single-Base Mismatches in Solution by Chemiluminescence”, Nucleic Acids Research, 56) References Cited 24(24):4998–5003 (1996). U.S. PATENT DOCUMENTS Nelson et al., “Simultaneous Detection of Multiple Nucleic 4,486,539 12/1984 Ranki et al.. Acid Targets in a Homogeneous Format, Biochemistry, 4,581,333 4/1986 Kourilsky et al.. 35(25):8429–8438 (1996). 4,683,195 7/1987 Mullis et al.. 4,751,177 6/1988 Stabinsky. Sambrook et al., Molecular Cloning A Laboratory 4,755,458 7/1988 Rabbani et al.. Manual, 2nd Ed., vol. 1, Chpt. 1, S1.90–1.91, “Plasmid 4,868,105 9/1989 Urdea et al.. Vectors'; and Chpt. 7, $7.37–7.57, “Extraction, Purification, 4,894,325 1/1990 Englehardt et al.. and Analysis of Messenger RNA from Eukaryotic Cells”. 5,112,734 5/1992 Kramer et al.. 5,124,246 6/1992 Urdea et al.. Sambrook et al., Molecular Cloning A Laboratory 5,130.238 7/1992 Malek et al.. Manual, 2nd Ed., vol. 2, Chpt. 9, $9.47-9.51, “Analysis and 5,200,314 4/1993 Urdea. Cloning of Eukaryotic Genomic DNA; Chpt. 10, 5,252,723 10/1993 Bhatt et al.. 5,273,882 12/1993 Snitman et al.. $10.2-10.70, “Preparation of Radiolabeled DNA and RNA 5,283,174 2/1994 Arnold, Jr. et al.. Probes'; and Chpt. 11, S11.47-11.57, “Synthetic Olignucle 5,288,609 2/1994 Englehardt et al.. otide Probes. 5,399,491 3/1995 Kacian et al.. 5,437,990 8/1995 Burg et al.. morrissey et al., Analytical Biochemistry 181 : 345-359 5,547,842 8/1996 Hogan et al.. (1989). 5,554,516 9/1996 Kacian et al.. 5,635,352 6/1997 Urdea et al. ................................ 435/6 5,656.207 8/1997 Woodhead et al.. Primary Examiner W. Gary Jones 5,658,737 8/1997 Nelson et al.. ASSistant Examiner Ethan Whisenant 5,702,896 12/1990 Collins et al. .............................. 435/6 Attorney, Agent, or Firm-Christine A. Gritzmacher; Carlos 5,731,148 3/1998 Becker et al.. A. Fisher 5,731,153 3/1998 Lucas et al.. 5,750,338 5/1998 Collins et al.. 57 ABSTRACT FOREIGN PATENT DOCUMENTS A method for capturing a target polynucleotide in a Sample 0159719 10/1985 European Pat. Off.. onto a Solid Support with an attached immobilized probe by 0265244 4/1988 European Pat. Off.. using a capture probe and two different hybridization 0328829 8/1989 European Pat. Off.. conditions, which preferably differ in temperature only, is 0370694 5/1990 European Pat. Off.. disclosed. The two hybridization conditions control the 0526912 2/1993 European Pat. Off.. order of hybridization, where the first hybridization condi 88O1302 2/1988 WIPO. tions allow hybridization of the capture probe to the target WO 89/10979 11/1989 WIPO. polynucleotide, and the Second hybridization conditions WO901.0716 9/1990 WIPO. allow hybridization of the capture probe to the immobilized WO 91/08307 6/1991 WIPO. probe. The method may be used to detect the presence of a 94.03472 2/1994 WIPO. target polynucleotide in a Sample by detecting the captured 9419023 9/1994 WIPO. 95.03430 2/1995 WIPO. target polynucleotide or amplified target polynucleotide. WO95/16055 6/1995 WIPO. 881.0315 12/1999 WIPO. 13 Claims, 4 Drawing Sheets U.S. Patent Aug. 29, 2000 Sheet 1 of 4 6,110,678 1 O 15 \ (a) ( (a) - , (e) FIG. 1A 1 O ? 2O N (a): -- to N-(a) F.G. 1B U.S. Patent Aug. 29, 2000 Sheet 2 of 4 6,110,678 22 1 O ) (A) Na A.F---. YP (-) A. (+) (c) S(a) | 23 (B) 24 - - - - - - - - - - - - - -op- - (+)O (C) (-) (D) --- $3 24 ! (E)E (+)(-) FIG. 2 U.S. Patent Aug. 29, 2000 Sheet 3 of 4 6,110,678 3O 10 35 31 33 1>(), - \ N(a) ? ( 34f -- 32 (c) N(a) (b) FIG. 3A 50 1 O 40 30 - ? \ N to v - Sof \ (c) '-(a) 34 (b) FIG. 3B 1 O 40 N (a) y (d)) / (a) 9 (b) FIG. 3C U.S. Patent Aug. 29, 2000 Sheet 4 of 4 6,110,678 (CI) (o) |8 ,G?VÝVOJLVOJLLVEÐJALOOVVJL£VOI?JLJLLVVVVVVVVVVVVVVÇ 18LLLLLLLLLLLLLL NINININININI*,G 18LLLLLLLLLLLLLL «»,«;•& • 18LLLLLLLLLLLLLL 0T 6,110,678 1 2 TWO-STEPHYBRIDIZATION AND labeled with Specific binding partners to immobilize primers CAPTURE OF A POLYNUCLEOTIDE and primer extension products. The label Specifically com plexes with its receptor which is bound to a Solid Support. RELATED APPLICATION SUMMARY OF THE INVENTION This application claims priority to U.S. Provisional Appli According to one aspect of the invention, there is dis cation No. 60/045,430, filed May 2, 1997, under 35 U.S.C. closed a method of capturing a target polynucleotide present S 119(e). in a Sample. The method includes the Steps of incubating a FIELD OF THE INVENTION mixture comprising a target polynucleotide, a capture probe, and an immobilized probe in a first hybridization condition The present invention relates to methods for capturing a that favors formation of a capture probe: target hybridization polynucleotide which may be present in a Sample onto a complex that includes the capture probe and the target Solid Support. The invention is particularly useful for Sepa polynucleotide, wherein the first hybridization condition rating a target polynucleotide from other material in a disfavors formation of an immobilized probe:capture probe Sample, and is preferably employed as part of a diagnostic 15 hybridization complex that includes the immobilized probe procedure to detect the presence of the target polynucleotide and the capture probe; and then incubating the mixture in a in a Sample. second hybridization condition that favors formation of the immobilized probe:capture probe hybridization complex, BACKGROUND OF THE INVENTION thereby capturing the target polynucleotide in an immobi A target polynucleotide is a polynucleotide present in a lized probe:capture probe: target hybridization complex that Sample that can be purified from one or more Sample includes the immobilized probe, the capture probe and the components and/or whose presence can be detected using target polynucleotide. In one embodiment, the first incubat different techniques. Such techniques are typically carried ing step uses a temperature below a T of the capture out as part of a diagnostic procedure to detect the presence probe: target hybridization complex and above a T of the of a target polynucleotide which is indicative of the presence 25 immobilization probe:capture probe hybridization complex, of an infectious agent or pathogenic condition. and the Second incubating Step uses a temperature below a The presence of a target polynucleotide base Sequence T of the immobilization probe:capture probe hybridization region, present in a target polynucleotide, can be detected by complex. Preferably, the Second incubating Step is achieved various methods Such as those using nucleic acid probes that by lowering the temperature of the first hybridization con hybridize to a target Sequence. Probes can be designed to dition by at least about 10 C., or by at least about 20° C. In detect different target Sequences Such as those characteristic one preferred embodiment the first incubating Step uses a of microorganisms, viruses, human genes, plant or animal temperature of about 60° C. and the Second incubating Step genes, and/or pathogenic conditions. uses a temperature of about 40 C. or lower. Alternatively, A technique for purifying a target polynucleotide, which the first incubating Step may use a Solution having a chemi is often used in diagnostic procedures, involves capturing a 35 cal Stringency that favors formation of the capture probe target polynucleotide onto a Solid Support. The Solid Support :target hybridization complex and disfavors formation of the retains the target polynucleotide during one or more Washing immobilization probe:capture probe hybridization complex, Steps of the target polynucleotide purification procedure. and the Second incubating Step lowers the chemical Strin Ranki et al., U.S. Pat. No. 4,486,539 describe a hybrid gency of the Solution, thereby favoring formation of the ization Sandwich technique for capturing and for detecting 40 immobilization probe:capture probe hybridization complex. the presence of a target polynucleotide. The technique One embodiment of the method also includes the step of involves the capture of the target polynucleotide by a probe purifying the immobilized probe:capture probe: target bound to a Solid Support and hybridization of a detection hybridization complex.