EP0253894A1 - Sonde d'adn et procede de preparation - Google Patents

Sonde d'adn et procede de preparation

Info

Publication number
EP0253894A1
EP0253894A1 EP87900291A EP87900291A EP0253894A1 EP 0253894 A1 EP0253894 A1 EP 0253894A1 EP 87900291 A EP87900291 A EP 87900291A EP 87900291 A EP87900291 A EP 87900291A EP 0253894 A1 EP0253894 A1 EP 0253894A1
Authority
EP
European Patent Office
Prior art keywords
dna
rna
single stranded
complementary
detected
Prior art date
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Withdrawn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
EP87900291A
Other languages
German (de)
English (en)
Inventor
Yasuo Murao
Shuntaro Hosaka
Kumiko Miura
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.)
Toray Industries Inc
Original Assignee
Toray Industries Inc
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.)
Filing date
Publication date
Application filed by Toray Industries Inc filed Critical Toray Industries Inc
Publication of EP0253894A1 publication Critical patent/EP0253894A1/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • This invention relates to a DNA probe which is used for detecting or quantifying a DNA or RNA originated from a virus, microorganism, plant or animal cell, or the like.
  • the base sequence of a DNA or RNA is unique to the virus or the organism containing the DNA or RNA.
  • the DNA or RNA hybridizes with a DNA or RNA which is complementary thereto to form a double strand.
  • DNA probes are used for detecting or quantifying DNAs and RNAs.
  • DNA probes are prepared by directly labelling a DNA or RNA complementary to a DNA or RNA of a virus, microorgaism or plant or animal cell to be detected with a label.
  • the most sensitive labels are radio labels.
  • the radio labels have the drawbacks in that the more sensitive, the shorter the half life period, that it is dangerous to handle, and that a special and expensive equipment is required.
  • Avidin is a basic protein with a molecular weight of 68,000 which is contained in egg white, and it has a high affinity with biotin with a molecular weight of 244, the affinity constant being as high as 10 15M-1
  • Labelling with an enzyme is conducted by labelling a DNA probe complementary to a DNA or RNA to be detected with biotin which does not hinder very much the hybridization of the DNA probe with the DNA or RNA to be detected because of its low molecular weight, and then binding an avidin-enzyme conjugation with the biotin on the DNA probe by means of avidin-biotin bond after the DNA probe is hybridized with the DNA or RNA to be detected.
  • the known methods for labelling a DNA with biotin includes nick translation method in which a nucleotide constituting the DNA is replaced with a biotin-conjugated nucleotide in the presence of deoxyribonuclease and DNA polymerase; and a method in which photobiotin (commercially available from BRESA Inc.) is reacted with DNA under irradiation of light. ' Antigen-antibody reaction is also utilized for labelling DNA probes.
  • the DNA probe is first labelled with a hapten such as biotin, fluorescein and N-acetoxy-2-acetylaminofluorene, and after the hybridization with a DNA or RNA to be detected, an antibody specific to the hapten bonded to the DNA probe, which antibody is labelled with an enzyme or a fluorescent substance, is complexed with the hapten on the DNA probe to detect the DNA or RNA to be detected.
  • a hapten such as biotin, fluorescein and N-acetoxy-2-acetylaminofluorene
  • the DNA probe when the DNA probe is hybridized with a DNA or RNA to be detected, the DNA probe must be denatured to single strand by an alkali or heat treatment. Further, since the DNA itself which is complementary to the DNA or RNA to be detected is labelled, the complementarity is degraded, so that the hybridization is interfered to decrease the detection sensitivity. Especially, if the DNA probe is directly labelled with a large molecular substance such as an enzyme, the hybridization is severely hindered. Further, a DNA or RNA originated from a source different from that of the DNA or RNA to be detected is often contaminated in the test sample.
  • the DNA region derived from the vector is not usually eliminated sufficiently. Therefore, if the test sample contains as a contaminant a DNA or RNA originated from the same source as the vector, the DNA or RNA is detected to bring about a false positive result.
  • the object of the present invention is to provide a DNA probe with a high detection sensitivity, which is safe to handle and which is simply used.
  • a DNA probe comprising a single stranded DNA fragment which is complementary to a DNA or RNA to be detected, and a double stranded DNA fragment having a non-radioactive marker or a functional group to which a non-radioactive marker can be attached.
  • the hybridization is not interfered by the label at all, so that the detection sensitivity is high. Further, since the region other than the DNA fragment which is subjected to the hybridization with the DNA or RNA to be detected is double stranded and does not hybridize with any DNA or RNA, even if a DNA or RNA of the same origin as the double stranded region of the DNA probe is contained in the test sample, the contaminant does not hybridize with the DNA probe, so that false positive result is not obtained.
  • the region of the DNA probe of the present invention which region is complementary to the DNA or RNA to be detected, is single stranded, there is no need to denature the probe before use, so that it is simply used. Since the DNA probe of the present invention does not utilize a radioactive label, the handling of the probe is safe and no special equipment is necessary. In cases where the double stranded region of the DNA probe of the present invention contains the functional group to which a non-radioactive marker can be attached, the DNA probe' can be directly labelled with an enzyme. This not only offers conveni-ence, but also makes it possible to identify an unknown DNA or RNA by using a mixture of the DNA probes of the present invention, each of which is labelled with a different marker.
  • Fig. 1 is a schematic view for explaining a method of preparing the DNA probe of the present invention
  • Fig. 2 is a schematic view for explaining another method of preparing the DNA probe of the present invention.
  • the DNA probe of the present inventxoii tia s a single stranded portion which is complementary to DNA or RNA to be detected.
  • the source of the DNA or RNA to be detected by the DNA probe of the present invention includes, for example, viruses such as hepatitis virus (A and B), AIDS virus (HIV-III), ATL virus (HIV—I), herpes simplex virus (type 1 and 2), cytomegalovirus, rubeola virus, rubella virus, poliovirus, coxsackie virus, echovirus, influenza virus, rabies virus, yellow fever virus, Japanese encephalitis virus, ffl ⁇ xburg disease virus, adenovirus, dengue virus, EB vi us * mumps virus, vaccinia virus, Parvovirus, Papovavirus, Rotavirus, Tanapoxvirus, Yabavirus, Lassa virus, tobacco mosaic virus; mycoplasmas; ricketts
  • viruses such as hepatitis virus (A and B), AIDS
  • the DNA or RNA to be detected may have the whole base sequence or have only a part of the sequence, and may be single stranded or double stranded.
  • the DNA fragment complementary to the DNA or RNA to be detected is usually one originated from the same source as the DNA or RNA to be detected. It should be noted however, any DNA or RNA including those extracted from the source virus, bacterium, microorganism or plant or animal cell; those produced by genetic engineering technique in which the DNA or RNA originated from the source is inserted in a vector and the vector is replicated in a host; and those chemically synthesized in cases where the base sequence of the DNA or RNA is known can be used.
  • the non-radioactive markers which may be used in the DNA probe of the present invention includes markers such as fluorescent substances, chemiluminescent substances and enzymes, and further includes substances which can combine such markers, such as low molecular weight substances including biotin and
  • N-acetoxy-2-acetylaminofluorene antibodies to which such low molecular weight substances act as haptens, high molecular weight substances such as avidin which can bind the low molecular weight substances, and conjugates of a marker and the above-mentioned substances.
  • the fluorescent substances include fluorescein and rhodamine.
  • Non-limiting examples of chemiluminescent substances include luminol, isoluminol, N-(4-aminobutyl)-N-ethyl isoluminol, N-(6-aminohexyl)-N-ethyl isoluminol, N-(4-aminobutyl)-N-ethylisoluminolhemisuccinamide, lophine, lucigenine, acridinium esters, pyrogallol, luciferin, indole, riboflavin, 2-methyl-6-phenyl-3,7-dihydroimidazo(1,2-a)-pyradine-3- one, and derivatives thereof.
  • Non-limiting examples of enzymes include peroxidase, beta-galactosidase, alkaline phosphatase and acid phosphatase.
  • the DNA may be directly labelled with a high molecular weight marker
  • a low molecular weight marker such as biotin and then to attach a high molecular weight substance such as avidin which specifically binds the low molecular weight substance, to which high molecular weight substance a marker such as an enzyme or a fluorescent substance is conjugated.
  • a marker such as an enzyme or a fluorescent substance is conjugated.
  • the functional groups to which a non-radioactive marker can be attached and known and non-limiting examples thereof include amino group, carboxyl group, mercapto group, hydroxyl group, epoxy group and formyl group. If the DNA has such a group, it can be directly labelled with an enzyme. How to incorporate such groups into DNA is described, for example, in European patent
  • a non-radioactive marker should be attached to the functional group.
  • the attachment of the non-radioactive marker to the functional group may be conducted before or after the hybridization with the DNA or RNA to be detected.
  • the double stranded region of the DNA probe of the present invention may be any DNA which is labelled with a non-radioactive marker or which has a functional group capable of attaching a non-radioactive marker, and to which a single stranded DNA complementary to the DNA or RNA to be detected can be ligated, and may be, for example, a vector DNA or a synthetic DNA.
  • a bacteriophage having a single stranded circular DNA such as 0X-174, S13, M12, fl, fd and M13 phages are preferred.
  • the size of the DNA probe of the present invention is not important and may widely vary from 12 bases to several tens kb.
  • the DNA probe of the present invention can be prepared by two fundamental methods.
  • a first single stranded DNA containing the fragment complementary to the DNA or RNA to be detected is hybridized with a second single stranded DNA which contains a region complementary to a portion of the first single stranded DNA, which portion is other than the DNA fragment complementary to the DNA or RNA to be detected, the second single stranded DNA having a non-radioactive marker or a functional group to which a non-radioactive marker can be attached.
  • a first single stranded DNA containing a single stranded DNA fragment complementary to the DNA or RNA to be detected is provided, and then a complementary DNA strand (hereinafter referred to as second DNA) is formed on a region of the first single stranded DNA, which region is other than the single stranded DNA fragment complementary to the DNA or RNA to be detected, using the region of the first single stranded DNA as a template, and using a nucleotide having a noa-radioactive marker or a functional group to which a non-radioactive marker can be attached.
  • the non-radioactive marker may be attached after the double stranded DNA is formed.
  • Phages i.e., viruses of which host is a bacterium or a ray fungus are known for long years.
  • phages 0X-174, S13, M12, fl, fd and M13 are known to have a single stranded circular DNA.
  • double stranded circular DNAs called as replication form are first formed, and then single stranded circular DNAs are formed using the double stranded circular DNA as a template, and the single stranded circular DNAs thus formed are then released from the cell in the form of a phage.
  • the preferred embodiment of the first method utilizes such a phage. First, double stranded circular DNA of the phage is taken from a host cells infected with the phage, and the double stranded circular DNA is then cut with a restriction enzyme to open the ring.
  • a double stranded DNA complementary to a DNA or RNA to be detected, which is cut with the same restriction enzyme is then recombined with the opened ring to form a double stranded circular DNA in which a DNA fragment complementary to the DNA or RNA to be detected is inserted (denoted by reference numeral 10 in Fig. 1).
  • the thus obtained double stranded circular DNA is then transfected to a host cell.
  • the double stranded circular DNA is replicated in the host cell, and a first single stranded circular DNAs 12 containing the DNA fragment complementary to the DNA or RNA to be detected are released from the host cell in the form of a phage.
  • a double stranded DNA (the DNA may be fragmented with a restriction enzyme, by ultrasonic treatment or by nick translation or the like) derived from the same phage is labelled with a non-radioactive marker 16 and then denatured to form a second single stranded DNA 18 which is complementary to the region of the first single stranded DNA, which region is other than the DNA fragment complementary to the DNA or RNA to be detected.
  • the DNA probe of the present invention may be obtained by hybridizing the first single stranded DNA 12 with the second single stranded DNA 18.
  • a functional group to which a non-radioactive marker can be attached may be introduced in the double stranded DNA 14 or in the single stranded DNA 18, and after hybridization, the non-radioactive markers may be attached to the functional group.
  • the second DNA is formed on the region of the first single stranded DNA, which region is other than the DNA fragment complementary to the DNA or RNA to be detected using the region of the first single stranded DNA as a template.
  • a nucleotide such as dUTP and dATP
  • a non-radioactive marker such as biotin, a hapten, fluorescent substance, chemiluminescent substance
  • the second DNA is formed in full or not can be confirmed by electrophoresis using as the control a separately produced standard DNA. If the formation of the second DNA cannot be completed using one primer, two or more primers may be hybridized with the first single stranded DNA.
  • the first single stranded DNA containing a fragment complementary to the DNA or RNA to be detected may be obtained, for example, as in the first method.
  • a synthetic DNA 24 is hybridized with a proper restriction site (EcoRI site in Fig.2) of the first DNA and at least one synthetic DNAs 22 as a primer is hybridized with the corresponding portions of the first single stranded DNA.
  • the portions of the first single stranded DNA to which the primers are hybridized must be in the region other than the fragment which is complementary to the DNA or RNA to be detected. Thereafter, the DNA is cut with the restriction enzyme.
  • a stopper which terminates the extension of the second chain must be placed at the restriction site instead of the synthetic DNA.
  • the primers 22 are extended using DNA polymerase in the presence of dUTP to which allylamine is bonded for the incorporation of an amino group, and in the presence of dATP, dCTP, dGTP and dTTP.
  • dATP dCTP
  • dGTP dGTP
  • dTTP dTTP
  • caproylamidobiotin-N-hydroxysuccinimide ester is reacted with the DNA to obtain a straight chain DNA probe of the present invention.
  • the DNA probe of the present invention may be used in the form of circle or in the form of straight chain.
  • the DNA probe of the present invention may be used in the same manner as the conventional DNA probes.
  • a test sample of a tissue, body fluid or the like which is suspected to contain a virus or a microoraganism of interest, or a test sample of a plant or animal cell or a cancer cell is fixed to a glass plate.
  • DNA or RNA extracted from the tissue, body fluid or the cell is fixed on a filter membrane of nitrocellulose or Nylon. Then the glass plate or the filter membrane is then incubated with the DNA or RNA to be detected which has been denatured to single strand.
  • the non-radioactive marker is attached to the functional group after hybridization to detect the hybridized probe. Washing is usually conducted in each step.
  • Example 1 The present invention may be better understood by referring to the following examples. It should be understood that the examples are presented for the illustration purpose only, and they should not be interpreted in any restrictive manner.
  • Example 1
  • the filter was then washed five times for 5 minutes each with a washing buffer (0.5 M NaCl, 0.5% Triton X-100, 1 mM EDTA, 2% BSA and 10 mM KP0 4 , pH6.5) and then washed twice for 2 minutes each with a pre-detection buffer (0.2 M sodium acetate, pH5.8).
  • the filter was incubated at room temperature for 15 hours in a solution which is a 100:1 mixture of a pre-detection buffer solution of 1 mM Naphthol AS-MX phosphate and a pre-detection buffer solution of 4 mg/ l of Fastviolet B salt.
  • the colored fractions were combined to obtain a biotin-labelled M13mpl9 RF DNA solution of about 1 jig/ml.
  • DNA Probe Solution (Hybridization Solution) A solution containing 300 ng/ml of M13mpl9 in which Ad2 DNA was inserted, 300 ng/ml of biotin-labelled M13mpl9 RF DNA which was denatured by being boiled for 5 minutes, 50% formamide, 4 x SSPE (0.72 M NaCl, 40 mM NaP0 4 , 4 mM EDTA, pH7.4) , 5 x Denhardt's solution (0.1% polyvinyl pyrrolidone 360, 0.1% Ficoll 400, 0.1% BSA), 0.1% SDS, 0.1 mg/ml denatured salmon sperm DNA and 10% dextran sulphate was incubated at 42°C for 16 hours.
  • 4 x SSPE (0.72 M NaCl, 40 mM NaP0 4 , 4 mM EDTA, pH7.4
  • 5 x Denhardt's solution (0.1% polyvinyl pyrrolidone 360, 0.1% Fi
  • Ad2 DNA purchased from BRL
  • a concentration of 1000 ng/ml 100 ng/ml, 10 ng/ml, 1 ng/ml and 0.1 ng/ml
  • the filter was heated at 80 C for 1 hour.
  • the filter was boiled in physiological saline for 10 minutes and was rapidly cooled, and was immersed and incubated in a pre-hybridization solution (50% formamide, 4 x SSPE, 5 x Denhardt*s solution, 0.1% SDS and 0.1 mg/ml denatured salmon sperm DNA) for 3 hours at 42°C.
  • a pre-hybridization solution 50% formamide, 4 x SSPE, 5 x Denhardt*s solution, 0.1% SDS and 0.1 mg/ml denatured salmon sperm DNA
  • the filter was then incubated in the previously prepared hybridization solution for 19 hours at 42 C, and was washed with 2 x SSC containing 0.1% SDS for 15 minutes at room temperature, twice with the same solution for 15 minutes at 60 C, and once with 2 x SSC which does not contain SDS at room temperature, and was immersed in a pre-detection buffer.
  • the spots on the filter were colored in the same manner as in the preparation of biotin-labelled M13mpl9 RF DNA, and the spots of 10 ng/ml or more of Ad2 DNA were detected.
  • the DNA probe solution were prepared in the same manner as in Example 1, except that the biotin-labelled M13mpl9 RF DNA was treated with a ultrasonicator (Kaijo Denki 4280) for 30 seconds at 1A.
  • Example 3 Preparation of M13mpl9 Single Stranded DNA in Which Hepatitis B Virus (HBV) DNA was Inserted (HB/M13)
  • One hundred microliters of an aqueous solution containing lug each of 5 kinds of synthetic oligo DNA of 15 bases, one of which being complementary to the Eco RI site of HB/M13, the other being complementary to eaqually spaced 4 portions of the M13 region of the HB/M13 was mixed with 40 ⁇ l of HB/M13 (0.5 j ⁇ g/jil) in TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH8.0) , and the mixture was incubated at 55 C for 5 minutes. Thereafter, MgCl 2 and NaCl were added to the mixture to final concentrations of 7 mM and 100 mM, respectively.
  • the DNA obtained in 1) was dissolved in 100 ⁇ ul of 0.1 M NaHC0 3 , and 20 ⁇ l of DMSO solution of ⁇ . ⁇ caproylamidobiotin-N-hydroxysuccinimide ester
  • a hybridization solution containing 500 ng/ml of the biotin-labelled DNA probe was prepared as in Example 1.
  • pBR322 vector on which HBV DNA is cloned by a conventional method was cleaved with restriction enzyme Sph I to open the ring, 5 ul aliquotes of the solution with concentrations of 1000 ng/ml, 100 ng/ml, 10 ng/ml and 1 ng/ml, respectively were spotted on a nitrocellulose filter.
  • the detection and quantification of HBV DNA which was inserted in pBR322 was conducted with a result that the spot of the concentration of 10 ng/ml or more was positive.

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

Abstract

Est décrite une sonde d'ADN présentant une sensibilité élevée et dont la manipulation est aisée. Ladite sonde comprend un fragment d'ADN à brin unique qui est complémentaire à un ADN ou ARN à détecter, et un fragment d'ADN à double brin présentant un marqueur non radio-actif ou un groupe fonctionnel auquel un marqueur non radio-actif peut être fixé.
EP87900291A 1985-12-27 1986-12-26 Sonde d'adn et procede de preparation Withdrawn EP0253894A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP292809/85 1985-12-27
JP29280985 1985-12-27

Publications (1)

Publication Number Publication Date
EP0253894A1 true EP0253894A1 (fr) 1988-01-27

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ID=17786625

Family Applications (1)

Application Number Title Priority Date Filing Date
EP87900291A Withdrawn EP0253894A1 (fr) 1985-12-27 1986-12-26 Sonde d'adn et procede de preparation

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EP (1) EP0253894A1 (fr)
WO (1) WO1987004165A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4900659A (en) * 1986-01-30 1990-02-13 Enzo Biochem, Inc. Nucleotide sequence composition and method for detection of Neisseria gonorrhoeae and method for screening for a nucleotide sequence that is specific for a genetically distinct group
US7090972B1 (en) 1986-11-24 2006-08-15 Gen-Probe Incorporated Methods for determining the presence of non-viral organisms in a sample
US7087742B1 (en) 1986-11-24 2006-08-08 Gen-Probe Incorporated Oligonucleotide probes for the detection and/or quantitation of non-viral organisms
KR880010128A (ko) * 1987-02-12 1988-10-07 나카사와 다로 단쇄 dna의 조제방법
US7172863B1 (en) 1988-12-09 2007-02-06 Gen-Probe Incorporated Nucleic acid probes and methods for detecting Neisseria gonorrhoeae
JP2646814B2 (ja) * 1990-08-07 1997-08-27 アイシン精機株式会社 核酸等の検定方法
US5424413A (en) * 1992-01-22 1995-06-13 Gen-Probe Incorporated Branched nucleic acid probes

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1231303A (fr) * 1983-08-05 1988-01-12 Robert J. Carrico Detection des bacteries par hybridation de l'acide nucleique
US4777129A (en) * 1983-12-12 1988-10-11 Molecular Diagnostics, Inc. Nucleic acid probe detectable by specific nucleic acid binding protein
GB8405437D0 (en) * 1984-03-01 1984-04-04 Amersham Int Plc Detecting polynucleotide sequences
IL75142A0 (en) * 1984-05-15 1985-09-29 Smithkline Beckman Corp Polynucleotide hybridization probes
CA1272443A (fr) * 1985-02-22 1990-08-07 Nanibhushan Dattagupta Essai de double hybridation en solution-phase pour deceler des sequences polynucleotidiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8704165A1 *

Also Published As

Publication number Publication date
WO1987004165A1 (fr) 1987-07-16

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