EP0183822A4 - Katalysierte nukleinsäurehybridisierung mittels enzymreagenz. - Google Patents

Katalysierte nukleinsäurehybridisierung mittels enzymreagenz.

Info

Publication number: EP0183822A4
Authority: EP; European Patent Office
Prior art keywords: carrier; micrograms; dna; hybridization; radioactive
Prior art date: 1984-06-01
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Withdrawn

Application number

EP19850903113

Other languages

English (en)

French (fr)

Other versions

EP0183822A1 (de

Inventor

Edward J Zapolski

Douglas M Gersten

Robert S Ledley

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

NATIONAL BIOMEDICAL RESEARCH FOUNDATION

NAT BIOMEDICAL RES FOUNDATION

Original Assignee

NATIONAL BIOMEDICAL RESEARCH FOUNDATION

NAT BIOMEDICAL RES FOUNDATION

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

1984-06-01

Filing date

1985-05-31

Publication date

1988-08-29

1985-05-31 Application filed by NATIONAL BIOMEDICAL RESEARCH FOUNDATION, NAT BIOMEDICAL RES FOUNDATION filed Critical NATIONAL BIOMEDICAL RESEARCH FOUNDATION

1986-06-11 Publication of EP0183822A1 publication Critical patent/EP0183822A1/de

1988-08-29 Publication of EP0183822A4 publication Critical patent/EP0183822A4/de

Status Withdrawn legal-status Critical Current

Links

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230000002255 enzymatic effect Effects 0.000 title claims description 15
238000007899 nucleic acid hybridization Methods 0.000 title description 3
108020004414 DNA Proteins 0.000 claims abstract description 75
238000009396 hybridization Methods 0.000 claims abstract description 50
238000000034 method Methods 0.000 claims abstract description 50
239000000523 sample Substances 0.000 claims abstract description 25
239000012634 fragment Substances 0.000 claims abstract description 23
239000011159 matrix material Substances 0.000 claims abstract description 17
102000001218 Rec A Recombinases Human genes 0.000 claims abstract description 16
108010055016 Rec A Recombinases Proteins 0.000 claims abstract description 16
102000052510 DNA-Binding Proteins Human genes 0.000 claims abstract description 14
108010025899 gelatin film Proteins 0.000 claims abstract description 11
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238000002105 Southern blotting Methods 0.000 claims abstract description 5
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238000001962 electrophoresis Methods 0.000 claims description 9
101710116602 DNA-Binding protein G5P Proteins 0.000 claims description 7
101710162453 Replication factor A Proteins 0.000 claims description 7
101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 claims description 7
101710176276 SSB protein Proteins 0.000 claims description 7
101710126859 Single-stranded DNA-binding protein Proteins 0.000 claims description 7
208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 claims description 7
206010042297 Subacute sclerosing panencephalitis Diseases 0.000 claims description 7
239000000243 solution Substances 0.000 claims description 7
ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 6
239000011521 glass Substances 0.000 claims description 6
239000007983 Tris buffer Substances 0.000 claims description 5
229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 5
ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 claims description 4
108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
229960001456 adenosine triphosphate Drugs 0.000 claims description 4
QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 claims description 4
238000013519 translation Methods 0.000 claims description 3
235000011178 triphosphate Nutrition 0.000 claims description 3
239000001226 triphosphate Substances 0.000 claims description 3
UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 3
TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical compound [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 claims description 2
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238000000926 separation method Methods 0.000 claims 1
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FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
239000011543 agarose gel Substances 0.000 description 5
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150000007523 nucleic acids Chemical class 0.000 description 4
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ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 3
241000588724 Escherichia coli Species 0.000 description 3
ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
230000008901 benefit Effects 0.000 description 3
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238000004925 denaturation Methods 0.000 description 3
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108020004707 nucleic acids Proteins 0.000 description 3
102000039446 nucleic acids Human genes 0.000 description 3
230000008569 process Effects 0.000 description 3
239000011780 sodium chloride Substances 0.000 description 3
102000053602 DNA Human genes 0.000 description 2
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238000009792 diffusion process Methods 0.000 description 2
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238000011065 in-situ storage Methods 0.000 description 2
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238000004153 renaturation Methods 0.000 description 2
239000000126 substance Substances 0.000 description 2
239000013077 target material Substances 0.000 description 2
108020004635 Complementary DNA Proteins 0.000 description 1
238000012270 DNA recombination Methods 0.000 description 1
230000004568 DNA-binding Effects 0.000 description 1
QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
108091027305 Heteroduplex Proteins 0.000 description 1
239000000020 Nitrocellulose Substances 0.000 description 1
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108020004682 Single-Stranded DNA Proteins 0.000 description 1
240000006365 Vitis vinifera Species 0.000 description 1
239000002253 acid Substances 0.000 description 1
238000013019 agitation Methods 0.000 description 1
239000003513 alkali Substances 0.000 description 1
239000012491 analyte Substances 0.000 description 1
238000013459 approach Methods 0.000 description 1
239000002585 base Substances 0.000 description 1
238000010804 cDNA synthesis Methods 0.000 description 1
238000006243 chemical reaction Methods 0.000 description 1
239000002299 complementary DNA Substances 0.000 description 1
LEOJDCQCOZOLTQ-UHFFFAOYSA-N dibutylcarbamothioyl n,n-dibutylcarbamodithioate Chemical compound CCCCN(CCCC)C(=S)SC(=S)N(CCCC)CCCC LEOJDCQCOZOLTQ-UHFFFAOYSA-N 0.000 description 1
230000029087 digestion Effects 0.000 description 1
238000001493 electron microscopy Methods 0.000 description 1
230000004927 fusion Effects 0.000 description 1
238000010353 genetic engineering Methods 0.000 description 1
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230000001404 mediated effect Effects 0.000 description 1
238000002844 melting Methods 0.000 description 1
230000008018 melting Effects 0.000 description 1
MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
229920001220 nitrocellulos Polymers 0.000 description 1
230000002285 radioactive effect Effects 0.000 description 1
238000005215 recombination Methods 0.000 description 1
230000006798 recombination Effects 0.000 description 1
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238000011160 research Methods 0.000 description 1
230000035945 sensitivity Effects 0.000 description 1
229910000162 sodium phosphate Inorganic materials 0.000 description 1

Classifications

- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6832—Enhancement of hybridisation reaction

Definitions

the invention generally relates to a hybridization method for treating target DNA fragments to identify known genetic sequences, and more particularly, a hybridization method which is catalyzed by use of an enzymatic hybridization reagent formed, inter alia, of RecA protein, single- stranded DNA binding protein (SSB) , and adenosine triphosphate (ATP) .
an enzymatic hybridization reagent formed, inter alia, of RecA protein, single- stranded DNA binding protein (SSB) , and adenosine triphosphate (ATP) .
Nucleic acid hybridization technology used to identify the presence of known genetic sequences in a particular sample, is a necessary element in many processes of modern biotechnology, and in almost all aspects of genetic engineering. Under appropriate conditions, single strand nucleic acids that are complementary (homologous) unite to form duplex strands. This process of hybridization is generally accomplished by utilizing conditions whereby duplex nucleic acid molecules unwind (“melt”) and slowly reanneal to natually form the classical double stranded helix. Because of the rigid specificity (only strands of complementary base sequence will pair) , the process is used to seek and identify regions of DNA (target material) that are complementary to a specific single strand of DNA which is employed as the probe.
DNA fragments are separated according to size by electrophoresis in agarose gel, denatured to single strands by treatment with alkali, and neutralized. They are then transferred to a membrane or to specially treated paper matrix by blotting. The transfer may be expedited -by electrophoresis. The nucleic acid fragments are then fixed to the matrix by baking.
the bound matrix containing the nucleic acids is then exposed to a prehybridization solution of noncomplementary DNA in order to mask extraneous DNA binding sites on the matrix, thereby reducing background.
Hybridization is then performed, and is accomplished by annealing the matrix bound target DNA with probe DNA in various formulations of media and at temperatures which can be as high as 65-70 ⁇ C. Some formulations contain up to 50% dimethylformamide, or other dangerous substances, to help lower the DNA melting temperature so that hybridization can occur by annealing.
the present invention generally relates to catalyzed hybridization utilizing an enzymatic hybridization reagent. More particularly, the invention relates to
⁇ a hybridization method for treating target DNA fragments to identify known genetic sequences the method involving the disposition of the target DNA fragments on a carrier, the formation of a hybridization reagent consisting of a radioactive- labelled probe DNA and catalyzing enzymes, the immersing of the carrier in the hybridization reagent for a given period of time, the washing of the carrier in at least one wash solution, the exposure of X-ray film to the carrier, and the developing of the X-ray film to detect and locate the radioactive- labelled probe DNA, thus identifying a known genetic sequence.
the present invention relates to the discovery of the fact that two proteins from the bacterium E. coli.
the RecA protein will pair homologous molecules of DNA by a mechanism that differs kinetically from the thermal reannealing of complementary DNA single strands. It will pair homologous single strands of DNA to form the duplex DNA, or it will promote the exchange of a single- strand DNA with duplex-strand DNA.
the SSB DNA binding protein binds cooperatively to single-strand regions of DNA to increase the conversion of single- strand DNA into double-strand DNA mediated by RecA protein.
both of these proteins have been used in the prior art to study the mechanisms of DNA recombination and repair.
the SSB DNA binding protein has been used in electron microscopy to locate single-strand regions of DNA.
biochemical activity of both of these proteins has been documented in the prior art, their use in DNA hybridization is unknown in the prior art.
the use of these two proteins when the target DNA is disposed on a matrix or carrier, rather than a solution, is also unknown in the prior art.
the subject invention describes a suitable, novel, rapid ' enzymatic alternative to physicochemical denaturation/renaturation for hybridization on a matrix, which alternative can bypass the fixation, denaturation, neutralization, lengthy incubation, high temperature and stringent washing steps of the prior art, as described previously.
the carrier or matrix comprises a gel film on which target DNA fragments are placed.
target DNA fragments can be transferred from such a matrix to another matrix by use of the Southern blotting technique, or variations thereof, and the hybridization method can proceed accordingly.
Target DNA Lambda DNA - Hind III digestion fragments (Bethesda Research Labs Inc., Gaithersburg, Maryland) were placed into sample wells (1.0 or 0.5 micrograms total DNA) , and were separated according to their molecular weight by horizontal electrophoresis in agarose gel (0.6% w/v) , affixed to a glass plate of 15 x 15 cm. in size, and the agarose gel was dried to provide target DNA.
Probe DNA Radioactive labelled probe DNA was prepared by nick translation of 1.0 micrograms lambda DNA using a reagent kit (BRL Inc. , Gaithersburg, Maryland) and 50 micro-Ci of deoxycytidine-5'-[ oc - 32 P] triphosphate (Amersham Corp., Arlington Heights, Illinois). The enzymatic hybridization reagent (5 milliliters thereof) was formed as follows: the probe DNA;
RecA protein 200 micrograms of RecA protein (P-L Biochemicals, Div. of Pharmacia, Inc., Milwaukee, Wisconsin) ;
SSB DNA binding protein 140 micrograms of SSB DNA binding protein (Worthington Diagnostic Systems, Inc. , Freehold, New Jersey) ;
Pentex Bovine Albumin 500 micrograms of Pentex Bovine Albumin (Miles Laboratories, Inc., Elkhart, Indiana);
the dried gel film (15 x 15cm) , containing the target DNA fragments, was immersed into a solution containing 0.5M NaOH, 3M NaCl for 15 minutes, then into 0.5M Tris, 0.3M NaCl for 15 minutes, and then into the hybridization reagent (5 ml) containing the labelled probe DNA, enzymes and co-factors. It was incubated at 37 ⁇ C for one hour. The glass plate bearing the film was washed by gentle agitation a total of 6 times for 5 mins. each wash.
SSPE includes (per liter) 8.7 gm. NaCl, 1.38 gm NaH 2 P0 4 • H 2 0, and 0.37 g .
EDTA diosodiu salt
ph 7.4 The location of 32 P-labelled probe DNA which was enzymatically hybridized to undenatured target DNA was determined by radiofluorography developed after 24 hours exposure to X-ray film at -70 ⁇ C.
the target DNA was transferred to nitrocellulose membrane by the Southern blotting technique and fixed by baking. Enzymatic hybridization was then performed exactly as described above (starting with immersion into the hybridization reagent) .
the .inventive technique presents the further advantage of employment of mild conditions; for example, incubation of the dried gel film and target DNA fragments, immersed in the hybridization reagent containing the labelled probe DNA> enzymes and co-factors, takes place at 37 ⁇ C. for one hour).
the hybridization method of the invention is characterized by increased sensitivity in that the enzymatic procedure employed therein is possibly more efficient than the thermal annealing technique employed in the prior art.

Landscapes

Chemical & Material Sciences (AREA)
Organic Chemistry (AREA)
Life Sciences & Earth Sciences (AREA)
Zoology (AREA)
Wood Science & Technology (AREA)
Proteomics, Peptides & Aminoacids (AREA)
Health & Medical Sciences (AREA)
Engineering & Computer Science (AREA)
Microbiology (AREA)
Biochemistry (AREA)
Physics & Mathematics (AREA)
Molecular Biology (AREA)
Biotechnology (AREA)
Biophysics (AREA)
Analytical Chemistry (AREA)
Immunology (AREA)
Bioinformatics & Cheminformatics (AREA)
General Engineering & Computer Science (AREA)
General Health & Medical Sciences (AREA)
Genetics & Genomics (AREA)
Chemical Kinetics & Catalysis (AREA)
Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Investigating Or Analysing Biological Materials (AREA)

EP19850903113 1984-06-01 1985-05-31 Katalysierte nukleinsäurehybridisierung mittels enzymreagenz. Withdrawn EP0183822A4 (de)

Applications Claiming Priority (2)

Application Number	Priority Date	Filing Date	Title
US61620584A	1984-06-01	1984-06-01
US616205		1984-06-01

Publications (2)

Publication Number	Publication Date
EP0183822A1 EP0183822A1 (de)	1986-06-11
EP0183822A4 true EP0183822A4 (de)	1988-08-29

Family

ID=24468452

Family Applications (1)

Application Number	Title	Priority Date	Filing Date
EP19850903113 Withdrawn EP0183822A4 (de)	1984-06-01	1985-05-31	Katalysierte nukleinsäurehybridisierung mittels enzymreagenz.

Country Status (3)

Country	Link
EP (1)	EP0183822A4 (de)
JP (1)	JPS61502282A (de)
WO (1)	WO1985005685A1 (de)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US4963658A (en) *	1987-09-04	1990-10-16	Molecular Devices Corporation	DNA detection method
US4978608A (en) *	1987-09-04	1990-12-18	Molecular Devices Corporation	DNA detection system
US6326136B1 (en)	1988-04-01	2001-12-04	Digene Corporation	Macromolecular conjugate made using unsaturated aldehydes
US5449603A (en) *	1989-10-24	1995-09-12	Stratagene	Method for hybridizing nucleic acids using single-stranded nucleic acid binding protein
US5605824A (en) *	1989-10-24	1997-02-25	Stratagene	Composition for hybridizing nucleic acids using single-stranded nucleic acid binding protein
FI941016A7 (fi) *	1990-05-07	1994-05-03	Daikin Indsutries Ltd	Kaksois-D-silmukan muodostumisen diagnostiset sovellutukset
US5273881A (en) *	1990-05-07	1993-12-28	Daikin Industries, Ltd.	Diagnostic applications of double D-loop formation
US5506098A (en) *	1991-09-04	1996-04-09	Daikin Industries, Ltd.	In situ hybridization method
WO1993015226A1 (en) *	1992-01-28	1993-08-05	President And Fellows Of Harvard College	Method for nucleic acid hybridization using single-stranded dna binding protein
WO1994003639A1 (en) *	1992-08-04	1994-02-17	United States Biochemical Corporation	NON-ISOTOPIC DETECTION OF NUCLEIC ACID SEQUENCES USING AN RecA LABEL
WO1995018236A1 (en) *	1993-12-28	1995-07-06	Daikin Industries, Ltd.	IN-SITU HYBRIDIZATION METHOD USING RecA PROTEIN AND RecA PROTEIN HAVING MARKER OR LIGAND FOR USE IN SAID METHOD
JP3093116B2 (ja) *	1994-09-30	2000-10-03	株式会社豊田中央研究所	核酸検出方法
SE9602062D0 (sv) *	1996-05-29	1996-05-29	Pharmacia Biotech Ab	Method for detection of mutations
US6287765B1 (en) *	1998-05-20	2001-09-11	Molecular Machines, Inc.	Methods for detecting and identifying single molecules
US7700281B2 (en)	2004-06-30	2010-04-20	Usb Corporation	Hot start nucleic acid amplification

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US4395486A (en) *	1981-08-19	1983-07-26	Medical College Of Ga. Research Inst., Inc.	Method for the direct analysis of sickle cell anemia

1985
- 1985-05-31 EP EP19850903113 patent/EP0183822A4/de not_active Withdrawn
- 1985-05-31 WO PCT/US1985/001026 patent/WO1985005685A1/en not_active Ceased
- 1985-05-31 JP JP60502635A patent/JPS61502282A/ja active Pending

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
No relevant documents have been disclosed. *
See also references of WO8505685A1 *

Also Published As

Publication number	Publication date
EP0183822A1 (de)	1986-06-11
WO1985005685A1 (en)	1985-12-19
JPS61502282A (ja)	1986-10-09

Legal Events

Date	Code	Title	Description
1986-04-26	PUAI	Public reference made under article 153(3) epc to a published international application that has entered the european phase	Free format text: ORIGINAL CODE: 0009012
1986-06-11	AK	Designated contracting states	Kind code of ref document: A1 Designated state(s): CH DE FR GB IT LI NL SE
1986-08-13	17P	Request for examination filed	Effective date: 19860607
1988-10-19	A4	Supplementary search report drawn up and despatched	Effective date: 19880829
1988-11-30	STAA	Information on the status of an ep patent application or granted ep patent	Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN
1989-01-18	18W	Application withdrawn	Withdrawal date: 19881028
2007-08-08	RIN1	Information on inventor provided before grant (corrected)	Inventor name: ZAPOLSKI, EDWARD, J. Inventor name: GERSTEN, DOUGLAS, M. Inventor name: LEDLEY, ROBERT, S.

Publication	Publication Date	Title
WO1985005685A1 (en)	1985-12-19	Catalyzed nucleic acid hybridization using enzymatic reagent
US5223414A (en)	1993-06-29	Process for nucleic acid hybridization and amplification
AU630087B2 (en)	1992-10-22	Method of amplifying and detecting nucleic acid sequences
US5348855A (en)	1994-09-20	Assay for nucleic acid sequences in an unpurified sample
US4446237A (en)	1984-05-01	Method for detection of a suspect viral deoxyribonucleic acid in an acellular biological fluid
EP0235726B1 (de)	1993-05-19	Schnellverfahren zum Nachweis von Nukleinsäure-Sequenzen in einer Probe mittels Probemarkierung
AU601021B2 (en)	1990-08-30	Assay for necleic acid sequences in a sample
US5028525A (en)	1991-07-02	Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ
Stanley et al.	1978	A different approach to RNA sequencing
US5985557A (en)	1999-11-16	Invasive cleavage of nucleic acids
US5830643A (en)	1998-11-03	Partially double-stranded oligonucleotide and method for forming oligonucleotide
EP2055781B1 (de)	2011-10-26	FEN-1-Endonucleasen, Mischungen und Spaltungsverfahren
US4486539A (en)	1984-12-04	Detection of microbial nucleic acids by a one-step sandwich hybridization test
US5376529A (en)	1994-12-27	Lactam-containing compositions and methods useful for the hybridization of nucleic acids
EP2287307B1 (de)	2014-04-09	Materialien und Verfahren zur Identifizierung und Analyse von DNA-Markern mit intermediären Tandem-Repeats
EP0455517A1 (de)	1991-11-06	Restriktionsamplifikationstestverfahren
EP0672177B1 (de)	2006-07-19	Verfahren fur nuklein saure hibridisierung mit verwendung von einzelstrangige dna bindende proteine
EP0800406A2 (de)	1997-10-15	Vorrichtung und verfahren zur plasmasterilisation
EP0610212B1 (de)	1996-05-15	Steigerung der polynukleotid hybridisierung
Brenner et al.	1970	Isolation and partial characterization of the transfer ribonucleic acid cistrons from Escherichia coli
JP2002514909A (ja)	2002-05-21	ハイブリダイゼーション特異性を増強するための組成物および方法
US6383756B1 (en)	2002-05-07	Method for non-radioactive detection of membrane-bonded nucleic acids and test kit
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