WO1992018650A1 - Detection d'adn/arn par polarisation de fluorescence - Google Patents

Detection d'adn/arn par polarisation de fluorescence Download PDF

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Publication number
WO1992018650A1
WO1992018650A1 PCT/US1992/002983 US9202983W WO9218650A1 WO 1992018650 A1 WO1992018650 A1 WO 1992018650A1 US 9202983 W US9202983 W US 9202983W WO 9218650 A1 WO9218650 A1 WO 9218650A1
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Prior art keywords
probe
nucleic acids
amplified
sequence
target
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PCT/US1992/002983
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English (en)
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Chao-Huei Jeffrey Wang
Harryl C. Ammons
Michael E. Jolley
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Baxter Diagnostics Inc.
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Priority to CA002084405A priority Critical patent/CA2084405A1/fr
Priority to AU16904/92A priority patent/AU656965B2/en
Publication of WO1992018650A1 publication Critical patent/WO1992018650A1/fr

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    • 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
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention relates to the field of detecting target nucleic acid sequences contained in complex biological mixtures. More particularly, the invention relates to detection of nucleic acid sequences originally present in such mixtures n extremely low concentrations, by first enzymatically amplifying the specific target sequences, and then detecting them in a substantially homogeneous assay utilizing fluorescence polarization. An increase in fluorescence polarization indicates the extent of hybridization of a probe with the amplified target sequences. Background of the Invention In he study of cell populations associated with disease states it is frequently found that only a subpopulation of available susceptible cells actually exhibit the morbid phenotype.
  • infectious diseases the proportions of cells which are passively or actively infected may be very low, and the disease caused by the infectious agent may go unnoticed clinically even though an infected individual can transmit the agent to others.
  • infectious agents are clammy, protozoans, certain bacteria, and many viruses.
  • viruses the human immunodeficiency virus
  • HIV HIV
  • Latency may extend to several years during which the infected individual is capable of transmitting the virus to others through intimate contact, sharing of intravenous injection apparatus, or through donation of blood products.
  • T cells thymus-derived lymphocytes
  • T cells possess highly specific HIV receptors on their surfaces to which the virus attaches to gain entry to the cell.
  • Monolingual antibodies grouped generally in the CD4 cluster, see Leukocyte Typing III, Ed. A.J. McMichael, Oxford University Press, 1987, and specific for the HIV receptor, have been isolated heretofore (see Kung et al., U.S. Patent No. 4,381,295).
  • a signal molecule can be attached to such antibodies which binds selectively to those cells expressing the receptor antigen thereby identifying the helper T cell subpopulation.
  • Quantitation of cell numbers of such lymphocyte subpopulations may conveniently be carried out in a flow cytometer.
  • helper cells In normal individuals approximately 50 percent of peripheral T cells are helper cells. In HIV infected patients, this proportion declines sharply because the virus is cytotoxic to helper T cells. In latent infections or early in the course of clinical disease, the proportion of the helper T cell population actually containing virus, in either lytic or latent phase, may be very low, even to the order of l to 1000. This means that in such patients, a 2 ml sample of blood may contain only one or a few copies of the virus or its genome. At his stage of infection, no antibodies to viral proteins can be detected, even with the most sensitive immunological techniques available. There is a great danger that individuals at such early stages of infection may transmit the virus in donated blood without the virus being detected by conventional screening methods.
  • immunoassays for detection of HIV have been described including enzyme- linked immunoassays (ELISA) , immunodiffusion assays, radioimmunoassays (RIA) , and the classical Western blot. Also a number of distinct assay strategies have been developed.
  • ELISA enzyme- linked immunoassays
  • RIA radioimmunoassays
  • One group of assays utilizes HIV viral antigens, particularly viral protein containing epitopes in conserved domains, bound covalently to a solid matrix. The matrix-bound enzyme is contacted with a serum sample, and any anti-antigen antibodies contained therein bind to antigen.
  • antiserum raised in a heterologou ⁇ species against human antibody antigens conjugated to an enzyme (ELISA) , fluorescent molecule, or other signal generating substance is then reacted with the washed matrix-bound antigen-anti-antigen complex.
  • the signal emitted by the signal generating substance is typically a chromophor, fluorescent signal, beta or gamma radiation, or other such measurable emission.
  • analysis of serum antibodies maybe obtained by Western blot consisting of gel electrophoresis of viral proteins, electrotransfer of the proteins to blotting paper, followed by reaction with antisera, and color development of the individual protein bands.
  • the Western blot analysis is employed on a confirming test by blood banks in blood screening procedures.
  • Another system utilizes a mixture of monoclonal antibodies conjugated to a signal generating substance, each individual antibody type being specific for a different structurally distinct epitope.
  • the theory is that a greater number of signal generating antibody molecules will bind to antigen if there are no overlapping specificities.
  • Another approach is to target nucleic acid sequences of the virus with a homologous nucleic acid probe coupled to a signal amplification system. Under renaturing conditions the viral RNA (or denatured DNA) anneals to the complementary sequence of an oligonucleotide probe. Detection of the hybrid is afforded by signal generating substances covalently conjugated to the probe.
  • the second major approach involves target amplification in which the target interacting with the signal-generating entity is itself multiplied in number. Since proteins cannot replicate, target amplification inherently requires a nucleic acid sequence, and an enzymatic system which can replicate the target sequence in vitro.
  • One such target amplification technique is disclosed in U.S. Patent Nos. 4,683,195 (Mullis et al.) and 4,683,202 (Mullis) , and is called polymerase chain reaction (PCR) amplification.
  • PCR polymerase chain reaction
  • Primers consisting of a oligonucleotide capable of mediating DNA synthesis from a single stranded template is added under conditions which favor annealing of the primers to their specific complementary sequences.
  • a thermostable DNA polymerase is added, and an extension reaction proceeds at 72°C in the presence of deoxynucleotide triphosphates, adenosine triphosphate and cofactors.
  • the reaction is run at high temperatures to avoid non-specific binding of primer to non-homologous sequences. Under these stringent conditions fidelity of polymerization to the desired sequences is very high. .
  • extension reaction is repeated. Such repetition of extension polymerization may occur several times until the target sequence is amplified in numbers sufficient to detect by any of the signal-conjugated probe assays described hereinabove.
  • a first primer oligonucleotide or oligonucleotide containing an RNA transcriptase promoter-binding sequence is annealed to the target sequence and extended by DNA polymerase or reverse transcriptase.
  • a second primer complementary to the newly formed oligomer in a region distal to the first primer binding sequence is added, annealed, and extended.
  • the resultant duplex DNA oligomer thus has a sequence flanking the target region and containing a transcriptional promoter.
  • RNA transcriptase in the presence of oligonucleotide triphosphates, adenosine triphosphate, and cofactors institutes transcription in vitro yielding up to 1000 copies of the target sequence.
  • the TAS methods have been disclosed in WO 88/01050 (Berg et al.).
  • RNAse H is added to the reaction mix.
  • RNAse H specifically catalyzes the step- wise hydrolysis of RNA bases in an RNA-DNA duplex, so that after a cDNA strand has been synthesized with reverse transcriptase the RNAse digests the RNA strand of the duplex to permit synthesis of the second complementary DNA strand by DNA polymerase. It will be apparent that since a heating step to melt the DNA-RNA duplex is unnecessary for cyclization of the reaction, the entire amplification can be performed in a single incubation.
  • the disadvantages of the TAS and 3SR methods, compared to PCR is the lesser degree of stringency because of non-specific primer bonding at the lower temperature.
  • target amplification can be carried out in a ligase-mediated procedure.
  • complementary primer sets form adjacent hybrids on both complementary strands of the target.
  • Ligase then joins the primers together at the nick separation, after hybridization.
  • the ligated double primer can then act as a template for further ligation of primers in a subsequent melting and rehybridiz tion step.
  • the nucleic acids are ordinarily extracted and the amplified sequences are detected by the procedures set forth hereinabove.
  • the known procedures of the prior art are heterogeneous, that is, they require multiple steps in which the DNA is first hybridized to a signal generating probe, followed by a step in which the unhybridized probe is separated from hybridized probe. Ordinarily different sets of reagents are required for signal generation than for probe hybridization and separation.
  • a completely homogeneous method for detection of amplified nucleic acids without a separation step is unknown in the prior art. Summary of the Invention
  • nucleic acids amplified by any method are detected in a convenient homogeneous one-step method utilizing fluorescence polarization.
  • the method of this invention comprises incubation of denatured amplified nucleic acids under hybridizing conditions with a nucleic acid probe of homologous sequence covalently coupled to a fluorophor.
  • the fluorophor-conjugated probe is added at a concentration sufficient to produce a measurable increase in fluorescence polarization when hybridized to the amplified nucleic acid sequences, but not in an excess quantity so as to appreciable quench the increase in fluorescence polarization attributable to hybridization.
  • the method of the present invention is adaptable to a simple automated format for convenient processing of a large number of samples. This is because there is no nucleic acid extraction step, and it is unnecessary to separate unhybridized probe from the mixture. Further, the increase in fluorescence polarization upon hybridization is virtually instantaneous, and no repetitious shifts in temperature are required in practicing the present method.
  • the method of the invention comprises: a) Heating or otherwise denaturing a liquid mixture and for a length of time sufficient to separate duplex nucleic acids into single strands, b) adding a fluorochrome-conjugated nucleic acid probe having a base sequence complementary to a target sequence contained in the amplified nucleic mixture, c) lowering the temperature of the mixture for adjusting the pH to permit hybridization of probe sequences to amplified nucleic acids target sequences; d) incubating the mixture for a time sufficient for substantially complete hybridization to occur; and e) measuring the degree of fluorescence polarization.
  • target nucleic acid sequences are incubated under hybridization conditions with a fluorescein-conjugated probe comprising an oligonucleotide having substantial complementarity to the target nucleic acid sequences, the fluorescein being amino-linked to the oligonucleotide probe through an aminochlorotriazinylaminoalkylphosphoryl group, incubating for a time sufficient to obtain substantially complete hybridization, and measuring the fluorescence polarization.
  • Probes for detecting a target nucleic acid sequence comprise a fluorescein-conjugated oligonucleotide sequence of substantial complementarity to the target nucleic acid sequence, the fluorescein molecule being amino-linked to the oligonucleotide portion through an aminochlorotriazinylaminoalkylphosphoryl group. Most preferred probes are selected from the group.
  • the method of claim 4 wherein the said probes are selected from the group consisting of oligonucleotides substantially homologous to target nucleic acids containing a guanosine or cytosine base in the position immediately 5' of the base annealing to the fluorescein-labelled terminal nucleotide of the said probe.
  • a kit is also contemplated by this invention, comprising a vessel containing a concentrated buffer solution of a composition optimizing hybridization of nucleic acids but without interfering with the measurement of fluorescence polarization, and a second vessel containing a solution of one or a plurality of nucleic acid probes conjugated to one or a plurality of assays.
  • the solutions of the kit of this invention are readily deliverable step-wise to a multiplicity of sample containers. The samples can be processed through each of the method steps without transferring to another container, so that the incubations and fluorescence polarization determination can be accomplished in the same vessel and same machine under automation.
  • fluorescence polarization In the technique of fluorescence polarization, light from a source is utilized to excite fluorescence emission from a fluorochrome molecule.
  • a fluorophor or fluorochrome is a molecular entity, usually of molecular weight less than 10,000 daltons which emits fluorescent light at a characteristic wavelength when impacted with a radiant energy source.
  • the fluorochrome is covalently coupled to a nucleic acid probe hybridizable with a complementary target amplified nucleic acid sequence.
  • fluorochromes are known in the art which are adaptable to the present invention, including fluorescein derivatives, acidine orange (Van Bertalanffy et al., J. Histochem. Cvtochem..
  • fluorescein is covalently attached to an oligonucleotide probe through an aminochlorotriazinylaminoalkyphosphoryl group havxng the generalized structure:
  • n is an integer from 2 to about 12 (hereinafter referred to as DTAF) .
  • DTAF fluorescein conjugted oligonucleotide amino-linked through an aminochlorotriazinylaminoethylphosphoryl group. It has been determined empirically that compounds of the preferred generalized structure are superior in generating signal than other amino-1inker configurations, such as carboxy-fluorescein succinimidyl ester or the fluorescein isothiocyanate derivative (FITC) having the structure:
  • oligonucleotide portion of the probe has a sequence of substantial complementarity to the amplified target nucleic acids, so that a duplex between probe and target is formed. It is apparent that perfect complementarity is not necessary so long as a stable duplex is formed under the hybridization conditions utilized, and that some base mismatching may be tolerated.
  • these probes may be used in the detection of any substantially homologous target nucleic acid sequence, and is not limited to detection of only amplified nucleic acid.
  • the principle of this technique is based upon the characteristic rotational properties of molecules in solution. Molecules have a tendency to tumbe about their various axes of rotation. In general, larger molecules tumbe more slowly than smaller ones.
  • the oligonucleotide fluorochro e-conjugated probes tumbe very rapidly and along several axes of rotation, fluorescent light emitted from such spinning molecules is diffuse and characteristically multiplanar.
  • the oligonucleotide portions of the probe may have a variable number of bases, preferably about 10-40 which are substantially homologous with the target sequence.
  • a variable number of bases preferably about 10-40 which are substantially homologous with the target sequence.
  • selection of conserved sequences is an important consideration because the envelope proteins-are genetically labile and mutant variations occur extremely rapidly. This has been found to result from replication errors. Reverse transcriptase is a highly imprecise replicator.
  • the sequence encoding the enzyme itself is highly conserved. Accordingly, the sequences encoding the reverse transcriptase make excellent oligonucleotide probes which can be expected to hybridize to virtually any HIV variant. Conversely, selection of probes of short hybridizable sequences, preferable of 6 to 12 bases, from highly mutogeneic genomic regions may be utilized diagnostically to assess the degree of drift in homology over a period of time, or between different patient hosts.
  • the method of the present invention is equally applicable t detection of the nucleic acid so amplified, without regard to the amplification method used. It will be apparent to those skilled in the art that the inventive method of detection will be applicable as well to any future improvement or new technology in the nucleic acid amplification art.
  • the buffer utilized during hybridization may be selected from the group of buffers having buffering agents with a pH in the range of 6.5 to 8, having an ionic strength of 100 500 mM, and having 10 to 100 M of a chelating substance.
  • a typical buffer (2Ox concentration) is described in Maniatis et al., Molecular Cloning. 1982 and contains 200 mM sodium monobasic phosphate, 3 M sodium chloride, 20 mM ethylene diamnetetracetic acid, at pH 7.4 It was found empirically that this buffer provides good conditions for hybridization without appreciable quenching or other deleterous effect on the fluorescence polarization detection step.
  • the kit of the present invention provides a first vessel in which amplification of specific nucleic acid is done in a small volume. The amplified nucleic acid is then denatured by boiling or by the addition of sodium hydroxide.
  • a second vessel of the kit contains the probe in a reconstitutable, lyopholozed, or concentrated liquid form which also contains hybridization buffer.
  • the second vessel of the kit is made of a substance which does not readily absorb oligonucleotide molecules to its surfaces. Examples of such materials are polyethylene, polypropylene, teflon, silanized or siliconized glass.
  • the opening of the vessels may have flange or valve means whereby to facilitate drawing of liquids contained therein into lines conveying the liquids to an automated machine for processing of a large number of amplified nucleic acid samples.
  • the target sequence selected for study was a section of the HIVPV22 sequence partially encoding reverse transcriptase of HIV.
  • a section of this sequence (bases 1195-2690) was cloned into plasmid p24L utilizing conventional techniques.
  • the oligonucleotide primers at the 3' and 5' termini are shown on the figure.
  • DNA polymerase preferably a highly purified form of recombinant enzyme from which the endonuclease encoding sequences have been deleted, is added to obtain chain extension. This procedure was repeated several times. This method of PCR amplification does not depart appreciably from the method disclosed in U.S. Patent Nos. 4,683,145 and 4,683,202, and variations reported in the literature.
  • telomeres Upon completion of amplification, he nucleic acids are once again denatured. Denaturation can be effected thermally by boiling or, preferably, by addition of NaOH at 55°C resulting in a pH of approximately 13. The fluorochrome-conjugated oligonucleotide probes are added. If denaturation was effected by boiling, renaturation will proceed by simply lowering the temperature to 37° - 48°C. If denaturation was effected at high pH, then a sufficient amount of Tris pH 5.5 buffer is added to reduce the pH to 8, so that hybridization of probe proceeds. Empirically, it was discovered that signal detection is enhanced when complementary probe pairs are employed, and also when more than one target sequence is used.
  • reagents comprised a probe designated DASH3-AMI-FITC which has the oligonucleotide sequence shown in Figure 2, covalently attached through an amide linkage to fluorescein isothiocyanate designated DASH3, and a second unconjugated oligonucleotide sequence complementary to DASH3-AMI-FITC, designated DASHIC, and a third unconjugated oligonucleotide sequence, designated DASH#, as a noncomplementary control.
  • DASH3-AMI-FITC which has the oligonucleotide sequence shown in Figure 2
  • DASHIC fluorescein isothiocyanate
  • DASH# a second unconjugated oligonucleotide sequence complementary to DASH3-AMI-FITC
  • DASH# unconjugated oligonucleotide sequence
  • the capture target sequence for labelled probe is a PCR amplified HIV target derived from amplification of the p24L plasmid known to contain the DASH sequence.
  • the results are shown in Figure 4 of the drawings. Hybridization was monitored over 55 minutes and carried not in a total reaction volume of 1.6 ml.
  • the glossary of graph points on the right side of the figure expresses the DNA content of the reaction mix as the number of tipomoles of pre-PCR DNA present prior to the amplification step and, correspond to 0, 10 2 , 10 3 , 10 4 , and 10 5 pre-amplification molecules of p24L plasmid DNA.
  • Denaturation was carried out by incubating 20 microliters PCR reaction mix with 25 microliters water and 5 microliters NaOH at 55°c for 15 minutes. Tubes containing 1.5 ml 5x SSPE buffer, 7 microliters of 3.2 M Tris-Hcl and 100 microliters DASH3-AMI-FITC (10 mM in 5x SSPE buffer) were prepared and background fluorescence polarization measured.
  • RNA fraction of H-9 cells infected with HIV virus was extracted by conventional methods. A portion containing 0.1 attomoles of HIV RNA was then amplified utilizing the 3SR amplification techniques described hereinabove. Test tubes for assay of probe-RNA hybrids were prepared by adding 1.5 ml 5x SSPE buffer to 100 microliters of a 10 mM DASH3-AMI-FITC solution. 3SR RNA was alkalie denatured. Sixty microliters of the denatured 3SR amplified RNA was added to the test tubes, and fluorescence polarization was monitored over a 45 minute time period.
  • the resolving power of the present method was evaluated.
  • the supernatant from a growing culture of H-9 cells infected with HIV was obtained and the DNA putatively contained therein was subjected to 60 cycles of PRC.
  • the amounts of supernatant amplified corresponded to a volume of cultured cells containing 10 2 , 10 3 , 10 4 cells.
  • identical control supernatants were prepared from growing cultures of non-infected H-9 cells.
  • Test tubes were prepared containing 1.5 ml 5x SSPE buffer, 5 microliters IN NaOH, 7 microliters 3.2 M Tris pH 5.5, and 100 microliters of 10 mM DASH3-AMI- FITC.
  • Target DNA was denatured by adding 5 microliters IN NaOH and, 25 microliters water to 20 microliters PCR reaction mix followed by incubation at 55°C for 15 minutes. Fifty microliters of the denatured PCR reaction solution was added to the test tubes containing the DASH3-AMI-FITC probe and hybridization proceeded with monitoring by fluorescence polarization.
  • Figure 6 shows that affirmative HIV sequence detection can be obtained of the PCR amplification of the supernatant containing as few as 200 cells. This means that the method of the present invention has sufficient sensitivity and resolving power to be useful as a routine serum screening assay for individuals with very early pre-clinical HIV infection.
  • the probe technology and hybridization conditions are critical.
  • the target sequences must have high binding affinity to probe and be highly specific for host sequences. Purity is also an important factor.
  • the fluorochrome-conjugated probe sequence length must be short enough that the rotational and relaxational properties show contrast in fluoroscence polarization values when hybridized. Specific probe sequence may influence the rate at which annealing of probe to target sequence occurs, compared to the rate at which reannealing of native complementary target regions occurs. While is does appear that the amino-linker probe coupling strategy utilized in these examples is particularly efficacious, p*" r linkage modes may theoretically be substituted by and error. le 5
  • the probe and target reagents were as follows: a probe designated DASH3-15-AMI-F ⁇ TC which has the oligonucleotide sequence shown in Figure 7 covalently attached through an amino-linker arm to fluorescein isothiocyanate, a second probe designated DASH3-15-AMI-DTAF whch has an identical sequence as the above probe but covalently attached through an amino- linker arm to dichlorotrianzinylaminofluorescein, and an unconjugated oligonucleotide target sequence designated T23-DASHIC which has the sequence shown in Figure 7 and which is complementary to both of the above probes.
  • each probe was added to the target sequence and fluorescence polarization was monitor as in Example 2.
  • the probe and target were as follows: a probe designated DASH3-15-AMI-DTAF which has the oligonucleotide sequence shown in Figure 8 covalently attached through an amino-linker arm to dichlorotrianzinylaminofluorescein, and five unconjugated oligonucleotide target sequence designated T23-DASHIC-G, T23-DASHIC-A, T23-DASHIC-T, and T23- DASHIC-C which have the sequences shown in Figure 8 and are complementary to the above probe.
  • the probe was added to each of target sequences and fluorescence polarization was monitored as in Example 2.

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Abstract

Procédé homogène de détection de séquences cibles amplifiées d'ADN ou d'ARN utilisant des sondes d'ADN ou d'ARN marquées par signal et présentant une polarisation de fluorescence amplifiée de façon détectable, quand elles sont hybridées à des séquences cibles. De ce fait, l'invention décrit un processus analytique approprié à une étape, ne nécessitant pas d'extraction d'acide nucléique ou d'étape de séparation de signal.
PCT/US1992/002983 1991-04-11 1992-04-10 Detection d'adn/arn par polarisation de fluorescence WO1992018650A1 (fr)

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CA002084405A CA2084405A1 (fr) 1991-04-11 1992-04-10 Detection de l'adn/arn par polarisation de fluorescence
AU16904/92A AU656965B2 (en) 1991-04-11 1992-04-10 Detection of DNA/RNA by fluorescence polarization

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US68392391A 1991-04-11 1991-04-11
US683,923 1991-04-11

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EP0678581A1 (fr) * 1994-04-18 1995-10-25 Becton, Dickinson and Company Détection d'amplification d'acide nucléique par polarisation de fluorescence
DE4418691A1 (de) * 1994-05-28 1996-02-22 Boehringer Mannheim Gmbh 3'-(4'-) nicht-radioaktiv markierte Nukleoside und Nukleotide mit Aminocarbonsäure-, Peptid- oder Carbonsäure-Spacer
US5593867A (en) * 1994-04-18 1997-01-14 Becton, Dickinson And Company Fluorerscence polarization detection of nucleic acid amplication
EP0774515A2 (fr) * 1995-11-15 1997-05-21 Becton, Dickinson and Company Détection d'acides nucléiques par polarisation de fluorescence
EP0774516A2 (fr) * 1995-11-15 1997-05-21 Becton, Dickinson and Company Détection d'amplification d'acide nucléique par polarisation de fluorescence
WO1997041257A1 (fr) * 1995-12-22 1997-11-06 Visible Genetics Inc. Procede, compositions et trousse de detection et d'identification de micro-organismes
EP0805870A1 (fr) * 1995-01-20 1997-11-12 NeXstar Pharmaceuticals, Inc. Ligands d'acide nucleique detectables par spectroscopie
EP0781853A3 (fr) * 1995-12-12 1997-11-19 Eli Lilly And Company Méthode de mesure d'acides nucléiques
WO1998026093A2 (fr) * 1996-12-13 1998-06-18 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Formation en epingle a cheveux d'analogue de nucleotides fluorescents pour la detection de l'hybridation d'acides nucleiques
US5786139A (en) * 1994-12-09 1998-07-28 Panvera Corporation Method and kit for detecting nucleic acid cleavage utilizing a covalently attached fluorescent tag
WO1998038334A1 (fr) * 1997-02-27 1998-09-03 Lorne Park Research, Inc. Dosage de nucleotides dans une solution a l'aide de sondes d'acides nucleiques peptidiques
US5846729A (en) * 1997-02-27 1998-12-08 Lorne Park Research, Inc. Assaying nucleotides in solution using a fluorescent intensity quenching effect
US6046004A (en) * 1997-02-27 2000-04-04 Lorne Park Research, Inc. Solution hybridization of nucleic acids with antisense probes having modified backbones
US6060242A (en) * 1997-02-27 2000-05-09 Lorne Park Research, Inc. PNA diagnostic methods
US6251591B1 (en) 1997-02-27 2001-06-26 Lorne Park Research, Inc. Quantitative method for detecting nucleotide concentration
US6255050B1 (en) 1998-05-22 2001-07-03 Lorne Park Research, Inc. Dynamic hybridization system
WO2001073118A2 (fr) * 2000-03-29 2001-10-04 Lgc (Teddington) Limited Balise d'hybridation et methode de detection et discrimination rapides de sequences
WO2002046453A2 (fr) * 2000-12-05 2002-06-13 Syngenta Participations Ag Procede et trousse d'identification d'enzymes de modification d'acides nucleiques et d'inhibiteurs de tels enzymes
US6413718B1 (en) 1996-05-01 2002-07-02 Visible Genetics Inc. Method for sequencing of nucleic acid polymers
US6503709B1 (en) 1997-07-03 2003-01-07 Id Biomedical Corporation Methods for rapidly detecting methicillin resistant staphylococci
US6589744B2 (en) 2001-11-26 2003-07-08 Syngenta Participations Ag Method and kit for identification for nucleic acid modification enzymes and inhibitors thereof
US6645733B1 (en) 1999-06-25 2003-11-11 Ingeneus Corporation Fluorescent intensity method for assaying binding between proteins or peptides
US7222059B2 (en) 2001-11-15 2007-05-22 Siemens Medical Solutions Diagnostics Electrophoretic trace simulator
CN110305969A (zh) * 2019-07-17 2019-10-08 合肥工业大学 基于荧光偏振检测掺假肉成分的方法

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Also Published As

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JPH05508323A (ja) 1993-11-25
EP0533906A4 (en) 1994-09-28
AU1690492A (en) 1992-11-17
CA2084405A1 (fr) 1992-10-12
EP0533906A1 (fr) 1993-03-31
AU656965B2 (en) 1995-02-23

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