WO1993010264A1 - Dosage par amplification par enzyme de restriction - Google Patents

Dosage par amplification par enzyme de restriction Download PDF

Info

Publication number
WO1993010264A1
WO1993010264A1 PCT/US1992/009606 US9209606W WO9310264A1 WO 1993010264 A1 WO1993010264 A1 WO 1993010264A1 US 9209606 W US9209606 W US 9209606W WO 9310264 A1 WO9310264 A1 WO 9310264A1
Authority
WO
WIPO (PCT)
Prior art keywords
oligonucleotide
tris
sequence
target
assay
Prior art date
Application number
PCT/US1992/009606
Other languages
English (en)
Inventor
Albert L. George, Jr.
Original Assignee
Oncor, 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 Oncor, Inc. filed Critical Oncor, Inc.
Priority to JP5509342A priority Critical patent/JPH07505760A/ja
Priority to EP92924356A priority patent/EP0620861A4/fr
Publication of WO1993010264A1 publication Critical patent/WO1993010264A1/fr

Links

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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • 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
    • C12Q1/6823—Release of bound markers
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]

Definitions

  • the present invention relates to methods, reagents and kits for the determination of the presence of target nucleotide sequences.
  • the present invention relates to a method for conducting an assay for the presence of a specific target nucleotide sequence in a sample containing an unknown quantity of said specific target nucleotide sequence and to probes for use in such an assay.
  • any gene or gene portion is, in essence, a specific sequence of nucleotide bases forming all or part of a polynucleotide molecule, it is possible to directly test the sample polynucleotide to discover if the specific sequence of nucleotide bases forming the gene is present in the sample.
  • Interest in specific sequences of nucleotide bases may involve detecting the presence of pathogens, determining the presence of alleles, detecting the presence of lesions in a host genome and detecting a particular mRNA or the modification of a cellular host, to name only a few illustrative examples.
  • Genetic diseases such as Huntington Chorea, muscular dystrophy, phenylketonuria, thalassemias and sickle cell anemia can be diagnosed by the analysis of an individual's DNA.
  • diagnosis or identification of viruses, viroids, bacteria, fungi, protozoa or any other plant or animal life form can be determined by hybridization assays with nucleotide probes. Nucleic acid detection assays of various types have been documented in the literature.
  • adenosine-uracil base pairs may additionally be formed by hydrogen bonding in DNA-RNA hybrid molecules.
  • nucleic acid hybridization Base pairing of nucleic acid strands for determination of the presence or absence of a given nucleotide sequence involving sample nucleotide sequences and a probe nucleotide sequence is commonly referred to as nucleic acid hybridization or simply hybridization.
  • the Southern blot is a well known method for transferring electrophoretically fractioned DNA from a gel matrix to a nitrocellulose solid support by passive diffusion, followed by hybridization to a labeled probe. Similar procedures are used for detecting RNA with minor modifications and this method is known in the art as the Northern blot. The use of dried agarose gels as the immobilized phase is known as the ⁇ nblot method. All of these assay techniques are valuable tools for analyzing mRNA's, clones, genes, fragments, flanking sequences, repetitive elements and the like.
  • U.S. Patent No. 4,358,535 describes a method for detecting pathogens using a target nucleic acid sequence. The method involves fixing a target nucleic acid sequence to an inert support before hybridization with a radioactively labeled nucleotide probe. The target nucleic acid sequence is then determined by detecting the presence of any label on the inert support.
  • European Patent Application No. 0 117 440 discloses non-radioactive chemically labeled polynucleotide probes and methods of using the probes.
  • the target nucleic acid sequence is also fixed to a solid support.
  • U.S. Patent Nos. 4,767,699 and 4,795,701 disclose nucleic acid displacement assays. These assays utilize two polynucleotides; one polynucleotide is labeled, and the other polynucleotide is used to displace the labeled probe from the target sequence, thereby allowing detection of the target molecule. These assays use ATP to detect whether hybridization has occurred with the target molecule. Many of the assays using nucleotide probes have problems in the detection systems. Sensitivity of the labeled probe and background levels that are generated during the assay often lead to erroneous results.
  • a target amplification method may be utilized.
  • This method known as PCR, is described in U.S. Patent No. 4,683,195 and uses a set of primers and a DNA polymerase to extend the nucleic acid sequence of the target nucleotide and amplify it for future probe detection. By amplifying the DNA sequence, the target nucleotide can be more efficiently detected with the nucleotide probe.
  • One of the problems encountered in this probe assay is contamination of the reaction medium.
  • ClassII restriction enzymes are known in the art for making double-stranded scissions at specific sites within a DNA molecule. These enzymes are prevalent in bacteria, contain only one type of subunit and Mg 2+ alone is required for DNA cleavage. DNA cleavage or scission occurs at specific sites within or adjacent to the enzyme's recognition site. More than 500 restriction enzymes have been isolated from bacterial strains to date. These restriction enzymes have been characterized primarily with respect to their recognition sequences and cleavage specificity. The majority of restriction enzymes or endonucleases recognize sequences 4-6 nucleotides in length, but some have been found with 7-8 base recognition sites.
  • recognition sites contain a dyad axis of symmetry and in most cases all the bases within the site are uniquely specified.
  • Recognition of the symmetrical sequence of the hybridized sequences or palindromes is made by endonucleases. Endonucleases with symmetrical recognition sites generally cleave symmetrically.
  • restriction enzymes with their specific cleavage sites is well recognized in the art. Usually restriction enzymes are used for the specific mapping, cloning and characterization of DNA sequences. However, they have been used in various nucleotide probe assays. For instance, U.S. Patent No. 4,683,194 discloses a method for detecting the presence or absence of a specific restriction site in a nucleic acid sequence by hybridization with a nucleic acid probe that is complementary to one strand of the nucleic acid sequence spanning the restriction site. The hybridized sequence is then cleaved with a restriction enzyme and the resulting cut and uncut oligomers are separated and detected based on the type of probe label.
  • U.S. Patent No. 4,876,187 Another type of assay that uses the concept of cleaving a nucleic acid sequence in a nucleotide probe is disclosed in U.S. Patent No. 4,876,187. This method is used to detect DNA or RNA sequences by specifically cleaving the nucleic acid sequence of the probe in at least one point thereby removing any reporter molecules not bound to a complementary target DNA sequence. This assay improves the signal to noise ratio of the detection system and is a highly sensitive assay.
  • the aforementioned assays do provide a method for detecting nucleic acid sequences in a target molecule, the need still exists for an assay system that provides very high sensitivity, ease of detection, less contamination in the assay medium and ease of operation, while avoiding false positive results.
  • an object of the present invention is to provide a highly sensitive nucleic acid recycling or probe amplification assay for detecting a nucleic acid sequence.
  • -Another object of the present invention is to provide an assay that recycles the target sequence of interest and thereby amplifies the labeled probe.
  • the present invention provides a method for detecting the presence of a nucleic acid sequence which contains a scissile linkage, that is cleavable by a cleaving enzyme, in a biological sample, said method comprising the steps of:
  • oligonucleotide which comprises a nucleic acid sequence and a scissile linkage that is substantially complementary to a nucleic acid target sequence, said oligonucleotide having a detectable marker attached thereto;
  • Another embodiment of the present invention recites: A method of detecting in a sample the presence of a nucleic acid sequence which contains a scissile linkage that is cleavable by a cleaving enzyme, said method comprising the steps of: (a) forming a reaction mixture by denaturing a target nucleic acid sequence containing a scissile linkage in the presence of an oligonucleotide which contains a scissile linkage and is substantially complementary to the nucleic acid sequence of the target molecule;
  • Still another embodiment of the present invention recites: A method for detecting the presence of a nucleic acid sequence which contains a scissile linkage that is cleavable by a cleaving enzyme, in a biological sample, said method comprising the steps of: (a) hybridizing a target molecule having a scissile linkage to a labeled oligonucleotide probe that has a complementary sequence to said target and a detectable marker to provide a probe:target duplex;
  • Another embodiment of the present invention recites: A method for cleaving a nucleic acid sequence said method comprising providing an oligonucleotide having a partially double-stranded recognition and cleavage site and adding to said oligonucleotide a sufficient amount of a ClassII restriction enzyme in a solvent that alters the dielectric constant of a reaction mixture and/or enhances cleavage.
  • Another object of the present invention is the use of a partial hairpin oligonucleotide sequence as the labeled oligonucleotide in the assay of the present invention using ClassII and ClassIIS restriction enzymes.
  • Yet another object of the presence invention is to provide kits for diagnosis of various diseases using the above-described method.
  • Fig. 2 is a schematic representation of the method of the present invention with a non-complementary non-target sequence.
  • Fig. 3 is an autoradiograph of several samples run using the second oligonucleotide according to the present invention.
  • Fig. 4 is a schematic representation of the method of the present invention using a partial hairpin oligonucleotide as the first oligonucleotide with a ClassIIS restriction enzyme.
  • Fig. 5 is a schematic representation of the method of the present invention using a labeled first oligonucleotide.
  • Fig. 6 is an autoradiograph of several samples run without using the second oligonucleotide according to the present invention.
  • Fig. 7 is a schematic representation of the method of the present invention using a partial hairpin oligonucleotide and a ClassII restriction enzyme.
  • the present invention relates to a method for detecting nucleic acid sequences of interest in a target molecule.
  • This assay may or may not use a second oligonucleotide that is able to reconstitute the scissile linkage of said target molecule, permitting cleavage of the first oligonucleotide, thereby recycling the target sequence and amplifying the amount of the cleaved first oligonucleotide probe. If the second oligonucleotide is not used to reconstitute the scissile linkage, the labeled oligonucleotide is still available for recycling the target sequence. It is advantageous to run this method without the use of the second oligonucleotide, since it is more cost effective.
  • oligonucleotide refers to a compound made up of the condensation of a small number of nucleotides. The exact size of the oligonucleotide will depend on many factors including the ultimate function of the use of the oligonucleotide.
  • scissile linkage refers to a site-specific recognition nucleotide sequence that is cleavable by the use of restriction enzymes or restriction endonucleases.
  • the terms “restriction endonucleases” and “restriction enzymes” refer to enzymes each of which cut double-stranded DNA at or near a specific recognition nucleotide site.
  • the term “partially double- stranded recognition and cleavage site” refers to a nucleotide sequence that is not fully double-stranded at the scissile linkage.
  • the term “partial hairpin sequence” refers to an oligonucleotide sequence that is able to partially hybridize to itself internally and has additional single-stranded nucleotide sequences that are complementary to the target sequence to be detected.
  • the sample nucleic acid which may be employed herein may be derived from any source(s), including organisms, provided that it contain either the particular restriction site of interest within a given nucleic acid sequence when using a ClassII restriction enzyme or can be cleaved downstream using a ClassIIS restriction enzyme.
  • the process may employ pure DNA which is single or double stranded or a cDNA or a mixture of nucleic acids.
  • Sources include, for example, plasmids such as pBR322, cloned DNA and genomic DNA from any source. Typical sources can be from biological samples including bacteria, yeasts, viruses, and higher organisms such as plants, birds, reptiles and mammals.
  • Genomic DNA may be prepared from blood, urine, tissue material such as chorionic villi or amniotic cells by a variety of techniques (such as those described by Maniatis et al., in Molecular Cloning, pp. 280-281 (1982) . If necessary or desired to lower the viscosity, the sample of prepared human DNA to be analyzed may be physically sheared or digested using a specific restriction endonuclease.
  • the first oligonucleotide used in the present invention is a single-stranded oligonucleotide and has a structure complementary to the nucleic acid sequence being detected.
  • the probe is usually DNA and may contain an unlimited number of bases. However, it is preferable that the probe contains up to about 100 bases, more preferably between about 10 to 40 bases.
  • the probe may be obtained from messenger RNA, from a complementary strand of cDNA obtained by reverse transcription of messenger RNA with reverse transcriptase or by cleavage of the genome, conveniently by endonuclease digestion, followed by cloning of the gene or gene fragment in accordance with known techniques. See, for example, Kornberg, DNA Replication. W.H.
  • the gene or DNA fragment may be used for preparation of the probe.
  • the probe may also be chemically synthesized using an automated synthesizer such as a MILLIGEN® synthesizer. Chemical synthesis of the probes is the preferred method to obtain the desired probe for use in the present invention.
  • the oligonucleotide probe will be labeled with a detectable marker using an atom, an inorganic radical, radionucleotide, heavy metal, antibody, enzyme, biotin, immunobio in and the like.
  • a radioactive label may be employed. Radioactive labels include 32 P, 3 H, 14 C and the like. Any radioactive label may be employed which provides for an adequate signal and has sufficient half-life.
  • Other labels include ligands, which can serve as a specific binding pair member to a labeled antibody, fluorescers, chemiluminescers, enzymes, antibodies which can serve as a specific binding pair member for a labeled ligand and the like.
  • labels have been employed in immunoassays which can be readily employed in the present assay.
  • the choice of label will be governed by the effect of the label on the rate of hybridization and binding of the probe to the genetic DNA. It will be necessary that the label provide sufficient sensitivity to detect the amount of DNA available for hybridization. Other considerations include ease of synthesis of the probe, readily available instrumentation, the ability to automate, convenience and the like.
  • the label is bound to the probe will vary depending upon the nature of the label.
  • a radioactive label a wide variety of techniques can be employed. Commonly employed is end labeling with a 7- 32 P-NTP and T4 polynucleotide kinase. Alternatively, nucleotides can be synthesized where one or more of the elements present are replaced with a radioactive isotope, e.g., hydrogen with tritium.
  • a terminal hydroxyl can be esterified with inorganic acids.
  • 32 P phosphate or 14 C organic acids can be esterified via the terminal hydroxy or esterified to provide linking groups to the label.
  • intermediate bases may be substituted with activatable linking groups which can be linked to the label.
  • Ligands and antiligands may be varied widely. Where a ligand has a natural receptor such as biotin, thyroxine and cortisol, the ligand can be used in conjunction with labeled naturally occurring receptors. Any compound can be used, either haptenic or antigenic, in combination with an antibody.
  • Enzymes of interest as labels will primarily be hydrolases, particularly esterases and glycosidases , phosphatases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone and the like.
  • Che iluminescers include luciferin and 2,3-dihydrophthalazinediones, i.e., luminol.
  • Yet another method for labeling and detecting the nucleotide probe is disclosed in U.S. Patent No. 4,868,103, in which an energy transfer results in the generation of bathochromic and/or delayed fluorescence emission.
  • Fluorescence radiation, emitted from a first energy emitter (El) is absorbed by a second energy emitter (E 2 ) .
  • the second emitter emits fluorescence radiation of a longer wavelength than the first energy emitter.
  • (E ⁇ and (E 2 ) must be within a proximate distance of each other so that the energy emitted by (E[) can be absorbed by (E 2 ) and (E 2 ) emits fluorescent energy at a longer wavelength.
  • any means for labeling and detecting the labeled probe can be used in the present invention. It is preferable, however, that the label is bound at a distance away from the scissile linkage and is situated at the 3 prime or 5 prime end of the first oligonucleotide.
  • the first oligonucleotide Besides having a label on the first oligonucleotide the first oligonucleotide must also contain a scissile linkage that is complementary to the target molecule of interest.
  • the scissile linkage must be readily cleavable by restriction enzymes or other means after hybridization has occurred when ClassII restriction enzymes are used.
  • restriction enzymes or other means after hybridization has occurred when ClassII restriction enzymes are used.
  • ClassIIS restriction enzymes requires the recognition sequence, but cleaves downstream from this sequence.
  • the first oligonucleotide is immobilized to a solid support.
  • a solid support in which an oligonucleotide can be affixed may be utilized in the present invention.
  • these supports include glass, test tubes, microtiter plates, nylon, beads, agarose beads, magnetic beads, glass beads, teflon, polystyrene beads, photodetectable chips and the like. It is well known within the art how to attach the oligonucleotides to a solid support by derivatizing one of the ends of the oligonucleotide with a carboxyl or amino group.
  • Another example of attachment is taking an avidin-bound bead and attach the oligonucleotide via biotin. If a solid support is used, it is advantageous to have the label at the opposite end of the oligonucleotide to which the support is attached.
  • the restriction enzymes are well known in the art and have specific recognition sites.
  • restriction enzymes that can be used in the present invention are ClassII restriction enzymes and include Aat II, Ace II, Ace III, Aha III, Alu I, Aoc I, Apa I, ApaL I, Ava I, Ava II, Bal I, Bam HI, Bel 1, Bgl II, BssH II, BstE II, Cla I, Dra I, Eco52 I, Eco RI, Eco RII, Eco RV, Fsp I, Hae II, Hha I, Hind III, Hpa I, Hpa II, Kpn I, K ⁇ p I, Nci I, M ⁇ t II, Nae I, Pst I, Pvu I, Xba I and the like.
  • restriction endonucleases there are more than 500 different restriction endonucleases which can be used and the present invention contemplates the use of any of these enzymes.
  • the criteria for choosing the restriction enzyme is based upon the recognition site of the target nucleotide molecule, the first oligonucleotide probe, and the properties of the enzyme.
  • the first oligonucleotide probe containing the scissile linkage must be complementary to the target nucleotide's recognition or scissile linkage site which upon hybridization of these oligonucleotides and addition of the restriction enzyme, the duplex will be cleaved.
  • an oligonucleotide containing a partial hairpin sequence may be utilized as the oligonucleotide, provided that this oligonucleotide is labeled.
  • the partial hairpin oligonucleotide sequence should be able to hybridize internally to itself thereby forming a loop and contains additional single-stranded oligonucleotide sequences that can hybridize to the target sequence of interest.
  • the hairpin loop can reside either at the 5 prime or 3 prime end of the oligonucleotide and is labeled appropriately as discussed above.
  • ClassIIS restriction endonucleases may be used to cleave double-stranded DNA at precise distances from their recognition sites.
  • ClassIIS restriction endonucleases include BJvI, BJvII, Binl, Fokl , Hgal , Hphl , MJboII, Mnll , SfaNI, Tagil, Tthlllll , HinGuI and the like. In particular, see for example, Szybalski, Gene, 40:169-173 (1985) and Podhajska et al Gene. 40:175-182 (1985).
  • ClassIIS restriction enzymes may also be used without partial hairpin oligonucleotide sequences in the practice of the present invention.
  • Figures 4 and 7 are illustrative of how the hairpin oligonucleotide can be used in the method of the present invention.
  • Restriction enzymes or endonucleases are relatively stable proteins. Their purification to homogeneity is often not necessary. All restriction enzymes require a cofactor for cleavage such as Mg 2+ or Mn 2+ and are most active in the pH range of 7.2 to 7.6. Typically for enzyme cleavage an appropriate buffer system is used. These buffer systems vary among the restriction enzymes used to cleave a hybridized duplex. Therefore, in addition to the DNA substrate and restriction enzyme, most reaction solutions will contain TRIS, i.e., tris(hydroxymethyl)aminomethane buffer, Mg 2+ , NaCl, 2-mercaptoethanol and bovine serum album (BSA) . The predominant difference among the restriction enzymes is their dependence on ionic strength. To maximize cleavage efficiencies the buffering systems are varied among the restriction enzymes. For instance, if a Hind III restriction enzyme is used in the assay, a buffer containing 50 mM NaCl, 25 mM
  • TRIS-HCl pH 7.7, 10 mM MgCl 2 , 10 mM ⁇ -mercaptoethanol and 100 ⁇ /ml BSA is used to maximize the cleavage efficiency. If an Eco RI enzyme is used the buffering system contains 50 mM NaCl, 100 mM TRIS-HCl, pH 7.5, 5 mM MgCl 2 and 100 ⁇ /ml BSA.
  • the present invention contemplates the use of different buffering systems, which vary according to the restriction enzyme used in the assay.
  • Restriction enzymes may also vary in temperature optima. Most cleavages are performed at 37°C, but a few endonucleases such as Sma I prefer lower incubation temperatures, and several, mainly those isolated from thermophiles such as Taq I, require much higher temperatures. Therefore, the reaction temperature in the present invention is chosen taking into consideration the restriction enzyme used in the assay.
  • any other method available can be used in the cleavage process. For instance, certain chemicals may be used to cleave doublestranded complexes at specific sites.
  • the present invention may also use a second oligonucleotide which reconstitutes with the cleaved target molecule of interest such that hybridization with the probe oligonucleotide occurs within the assay.
  • the second oligonucleotide acts to recycle the target sequence of interest and thereby amplifies the amount of cleaved probe for detection purposes.
  • This second oligonucleotide is a single-stranded nucleotide that is complementary to an end of the first probe oligonucleotide such as the 3 prime end or 5 prime end.
  • the second oligonucleotide may be derived via processes similar to the synthesis of the first oligonucleotide, as described above.
  • the second oligonucleotide should contain bases such as a 3 prime or 5 prime base that will reconstitute the target molecule at the site that was cleaved by the restriction enzyme. After reconstituting with the cleaved end of the target molecule, hybridization once again occurs with the first labeled oligonucleotide and the process "cycles" once again and releases the labeled probe into the reaction media.
  • the second oligonucleotide may contain up to 100 bases, preferably between about 10 to 20 bases, more preferably about 14 bases.
  • Figures 1 and 2 are illustrative examples of how the present invention works.
  • the second oligonucleotide is not utilized and therefore reconstitution of the cleaved end of the target molecule does not occur. Even though a second oligonucleotide may not be used in the preferred embodiment of the present invention, nonetheless, the cleaved detectable marker does accumulate. Thus, positive target sequences are easily detectable. Contrary to teachings in the prior art, it has been discovered by the present inventor that the use of ClassII restriction enzymes requires only a partially double-stranded recognition and cleavage site in order to cleave the hybridized oligonucleotide under varying chemical conditions.
  • Examples of the various chemicals (which are generally diluted in appropriate solvents) that can be used to enhance the cleavage of the partially double-stranded recognition and cleavage site in the present invention include divalent cations such as cobalt, magnesium, zinc and the like, glycerine, di ethylsulfoxide (DMSO) , dimethylformamide (DMF) , formamide, ethylene glycol and the like. See, George et al. Journal of Biological Chemistr , 255. pp. 6521-6524 (1980) .
  • the concentrations of these diluted chemicals, that may be used to vary the dielectric constant and/or enhance cleavage of the restriction site may vary depending on the assay conditions. Usually between 10% to 50% by volume of the aforementioned chemicals may be used; preferably between 10% to 30% by volume.
  • Figures 5 and 6 are illustrative examples of how the present invention works without the use of the second oligonucleotide.
  • the assay is initiated by denaturing the sample target molecule to form a single-stranded molecule.
  • the denaturation of the target molecule or substrate is generally performed by boiling.
  • the substrate, first oligonucleotide, second oligonucleotide buffer and distilled water may be mixed together and boiled for 5 to 10 minutes. The mixture is then allowed to cool to room temperature. After cooling, the restriction enzyme may then be added to initiate the reaction.
  • the procedure differs from that described above. Basically, the reaction requires that the ClassII restriction enzyme be cut initially in the reaction mixture comprising the labeled oligonucleotide, then the reaction mixture is denatured, hybridized and then recut again.
  • Figure 7 illustrates the use of ClassII restriction enzymes using a partial hairpin oligonucleotide.
  • the temperature at which the assay is run may vary according to the restriction enzyme used, the length of the oligonucleotide probe and the G + C content of the oligonucleotides present in the assay.
  • This empirical formula can be used to estimate the reaction temperature, but one must also take into account the restriction enzyme used in the assay. As discussed above, many restriction enzymes are active at 37°C, but others may require higher or lower temperatures.
  • the temperature of the reaction is chosen such that optimal rates of hybridization, as well as cleavage of the restriction enzyme occurs.
  • the reaction time may vary depending upon the concentration of the sequence of interest, the stringency, the length of the complementary first oligonucleotide probe sequence, the restriction enzyme used and the like. Enough time should be provided to permit amplification of the probe by recycling the target sequence of interest. Usually the assay is run from 1 to 3 hours, more preferably for two hours.
  • reaction After the reaction has run, it is stopped by placing an aliquot of the reaction mixture into a polyacrylamide gel loading buffer. The sample is then electrophoresed and an autoradiograph is taken of the electrophoresed gel. One can then proceed to quantitate the amount of probe generated on a scanning densitometer, if desired.
  • Two oligonucleotides may be used in the restriction amplification process.
  • the synthesis of the first oligonucleotide having the sequence 5' ACC ATG GCT GAT CCT GCA GGT ACC AAT G 3' (28 mer) and the second oligonucleotide having the sequence 5' GGA TCA GCC ATG GT 3 ' (18mer) were prepared by phosphoramidite chemistry and were subsequently purified by anion exchange HPLC.
  • the oligonucleotides synthesized by the phosphoramidite approach contained a free 5'-OH.
  • the partial hairpin oligonucleotide can be synthesized in the same manner as described above.
  • the first oligonucleotide contained a 5'-OH group which was labeled by transfer of the [ 32 P] hosphate from [T ⁇ A P using polynucleotide kinase.
  • the labeling reaction was carried out by dissolving each oligonucleotide in distilled water. The concentration of each oligonucleotide dissolved in the water was 0.5 x 10" 5 molar.
  • the reaction was performed using 1 ⁇ l of the first oligonucleotide (0.5 x 10 "5 molar), 5 ⁇ l of kinase buffer which contained 0.5 M TRIS-HCl at pH 7.6, 1 mM spermidine, 50 mM dithiothreitol, 1 mM EDTA and 0.1 M MgCl 2 , 30.5 ml distilled water, 12.5 ⁇ l 32 P-ATP (0.125 millicuries or 3000 curies/ ⁇ mole) and 1 ⁇ l of polynucleotide kinase (10 units) .
  • the above-mentioned reaction ingredients were combined and incubated at 37°C for l hour. The reaction was terminated by heating the reacted mixture to 80°C for 10 minutes.
  • the partial hairpin oligonucleotide can be labeled in the same manner as the first oligonucleotide.
  • the labeled first oligonucleotide and partial hairpin oligonucleotide prepared by this method are stable for at least one week when stored at -20°C.
  • HPV 16 Substrate The HPV16-DNA virus was isolated from a cervical lesion by methods known in the art and cloned into pBR322 using standard techniques. The cloned DNA was isolated by a modified procedure described by H.C. Birnboim and J. Daly, Nucleic Acids Res.. 7, 1513 (1979). The cells were grown overnight in 5 ml of
  • LB broth containing 100 ⁇ g/ml ampicillin at 37°C with vigorous shaking 1.5 ml of the culture was transferred to a 1.5 ml centrifuge tube and centrifuged at 10,000 X g for 1 minute. The supernatant was then carefully removed leaving the pellet as dry as possible.
  • the cells were resuspended by vortexing in 100 ⁇ l of an ice-cold solution containing 50 M glucose, 10 mM EDTA and 25 mM TRIS-HCL at pH 8.0. The resuspended cells were allowed to incubate for 5 minutes at room temperature.
  • the mixture was then centrifuged for 5 minutes at 10,000 X g, and the supernatant was transferred to another tube. The supernatant was centrifuged again at 10,000 X g for 5 minutes, and the supernatant was transferred to another fresh test tube. RNase A was then added to the supernatant, having a final concentration of 20 ⁇ g/ml. The reaction was incubated at 37°C for 20 minutes. After incubation, an equal volume of phenol: chloroform (1:1, saturated with 50 mM TRIS-HCL at pH 8.0, 100 mM NaCl, 1 mM EDTA) was added and the mixture was vortexed for 30 seconds and centrifuged at 10,000 g for 30 seconds. The aqueous phase was then transferred to a fresh test tube.
  • the restriction amplification assay was initiated by combining the HPV 16 substrate, the first oligonucleotide labeled probe, the first unlabeled oligonucleotide, a Pst I buffer, and deionized water. In this RAMP assay two controls were simultaneously run with the samples. Two different temperatures of 32°C and 37°C were utilized in this example.
  • Each reaction assay contained 1 ⁇ l (100 ng) of HPV 16, 2 ⁇ l of the labeled first oligonucleotide, 2 ⁇ l of unlabeled first oligonucleotide, 4 ⁇ l of the second oligonucleotide, 5 ⁇ l of Pst I buffer containing 100 mM NaCl, 10 mM TRIS-HCl at pH 7.7, 10 mM MgCl 2 , 1 mM DTT and 100 ⁇ g/ml BSA. 5 ⁇ l of Pst I was used. Distilled water was added in varying quantities such that the final volume in each assay tube was equal to 50 ⁇ l.
  • the first tube (#1) contained 2 ⁇ l of the first oligonucleotide labeled with 3 P, 2 ⁇ l of cold unlabeled first oligonucleotide, 4 ⁇ l of the second oligonucleotide, 5 ⁇ l Pst I buffer and 32 ⁇ l distilled water. No HPV 16 was added to the first assay mixture.
  • the second assay tube (#2) contained 1 ⁇ l (100 ⁇ g) HPV-16, 2 ⁇ l of the first oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled first nucleotide, 4 ⁇ l of the second oligonucleotide, 5 ⁇ l of Pst I buffer and 31 ⁇ l of distilled water.
  • the third assay tube (#3) contained 1 ⁇ l of HPV 16 (100 ⁇ g) , 2 ⁇ l of the first 32 P oligonucleotide, 2 ⁇ l of cold first oligonucleotide, 5 ⁇ l of Pst I buffer, and 35 ⁇ l of distilled water.
  • the fourth assay tube (#4) contained 1 ⁇ l of HPV 16 (100 ⁇ g) , 2 ⁇ l of the first 32 P oligonucleotide, 2 ⁇ l of cold unlabeled first oligonucleotide, 5 ⁇ l of Pst I buffer, and 35 ⁇ l of distilled water.
  • the fifth assay tube (#5) contained 1 ⁇ l of HPV 16 (100 ⁇ g) , 2 ⁇ l of labeled 32 P first oligonucleotide, 2 ⁇ l of cold unlabeled first oligonucleotide, 4 ⁇ l of second oligonucleotide, 5 ⁇ l of Pst I buffer, and 31 ⁇ l of distilled water.
  • the five assay tubes described above Prior to addition of Pst I, the five assay tubes described above were boiled for 5 to 10 minutes and then cooled at room temperature for approximately 10 minutes. After cooling the samples, the samples were placed in an incubator; assay tube numbers l, 4, and 5, discussed above, were incubated at 37°C. Assay tubes numbers 2 and 3 were incubated at 32°C. To initiate the assay, 5 ⁇ l of Pst I enzyme was added to each assay tube. The assay was run for 2 hours.
  • reaction was stopped by adding 10 ⁇ l of a solution containing 80% formamide, 15 M TRIS-HCl at pH 7.6, 1 mM EDTA, 0.1% w/v bromophenol blue and 0.1% w/v xylene cyanole FF to each assay tube. Each tube was then heated for 5 minutes at 65°C and cooled rapidly.
  • Polyacrylamide gels were prepared by making a stock solution containing 30% acrylamide (19:1 acrylamide:bis) , 19 grams of acrylamide, 1 gram of N,N'-methylenebisacrylamide and enough deionized water to dilute the solution to 67 ml total volume.
  • a concentrated solution of TBE buffer was prepared by diluting to 1 liter, 108 grams of TRIS base, 55 grams of boric acid and 9.3 grams of Disodium EDTA-2 ⁇ 0. The pH of the concentrated TBE buffer should be adjusted to 8.3, if appropriate.
  • 48 grams of ultra-pure grade urea (8 M) was added to the stock solution containing the 30% acrylamide. 10 ml of TBE buffer was added and the urea was dissolved in this solution.
  • the gels were then wrapped in a plastic folder placed next to Kodak X-OMAT AR film and exposed for 10 minutes.
  • Figure 3 is an illustrative example of the autoradiograph of the present invention. From the autoradiograph, one can easily determine the formation of the substrate by the presence of the 28 mer oligonucleotide band and the 18 mer oligonucleotide band. Lanes 2 and 5 illustrate a doublet pattern when the substrate is present in the reaction media at two different temperatures. Lanes 3 and 4 are indicative of the presence of only the labeled first oligonucleotides without the second oligonucleotide used to recycle the substrate. Lane 1 is indicative of the pattern obtained when both the first and second oligonucleotides are present in the assay mixture, but no substrate (i.e., HPV 16) was present in the reaction.
  • substrate i.e., HPV 16
  • restriction amplification assay Using One Oligonucleotide The restriction amplification assay was initiated by combining the HPV 16 substrate, the first oligonucleotide labeled probe, Pst I buffer, deionized water and varied concentrations of glycerine. In this RAMP assay, a control was run with 5% glycerine.
  • Each reaction assay contained 1 ⁇ l (100 ng) of HPV 16, 2 ⁇ l of the labeled first oligonucleotide, 2 ⁇ l of unlabeled first oligonucleotide, 5 ⁇ l of Pst I buffer containing 100 mM NaCl, 10 mM TRIS-HCl at pH 7.7, 10 mM MgCl 2 , 1 mM DTT and 100 ⁇ g/ml BSA. 5 ⁇ l of Pst I was used. Distilled water was added in varying quantities such that the final volume in each assay tube was equal to 50 ⁇ l, after addition of the glycerine in varied quantities.
  • the first tube (#1) contained 2 ⁇ l of the first oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled first oligonucleotide, 5 ⁇ l Pst I buffer, 1 ⁇ l HPV 16, 2.5 ⁇ l glycerine (5% by volume glycerine) and 32.5 ⁇ l distilled water. Tube #1 was used as a control.
  • the second assay tube (#2) contained 1 ⁇ l (100 ⁇ g) HPV-16, 2 ⁇ l of the first oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled first nucleotide, 5 ⁇ l of Pst I buffer, 5 ⁇ l glycerine (10% by volume glycerine) and 30 ⁇ l of distilled water.
  • the third assay tube (#3) contained 1 ⁇ l of
  • HPV 16 (100 ⁇ g) , 2 ⁇ l of the first 32 P oligonucleotide, 2 ⁇ l of cold first oligonucleotide, 5 ⁇ l of Pst I buffer, 10 ⁇ l glycerine (20% by volume glycerine) and 25 ⁇ l of distilled water.
  • the fourth assay tube (#4) contained 1 ⁇ l of HPV 16 (100 ⁇ g) , 2 ⁇ l of the first 32 P oligonucleotide, 2 ⁇ l of cold unlabeled first oligonucleotide, 5 ⁇ l of Pst I buffer, 15 ⁇ l glycerine (30% by volume glycerine) and 20 ⁇ l of distilled water.
  • the fifth assay tube (#5) contained 1 ⁇ l of HPV 16 (100 ⁇ g) , 2 ⁇ l of labeled 32 P first oligonucleotide, 2 ⁇ l of cold unlabeled first oligonucleotide, 5 ⁇ l of Pst I buffer, 20 ⁇ l glycerine (40% by volume glycerine) and 15 ⁇ l of distilled water.
  • the sixth assay tube (#6) contained 1 ⁇ l of HPV 16 (100 ⁇ g) , 2 ⁇ l of the first 3 P oligonucleotide, 2 ⁇ l of cold unlabeled first oligonucleotide, 5 ⁇ l of Pst I buffer,
  • reaction was stopped by adding 10 ⁇ l of a solution containing 80% formamide, 15 mM TRIS-HCl at pH 7.6, 1 mM EDTA, 0.1% w/v bromophenol blue and 0.1% w/v xylene cyanole FF to each assay tube. Each tube was then heated for 5 minutes at 65°C and cooled rapidly.
  • Polyacrylamide gels were prepared by making a stock solution containing 30% acrylamide (19:1 acrylamide:bis) , 19 grams of acrylamide, 1 gram of N,N'-methylenebisacrylamide and enough deionized water to dilute the solution to 67 ml total volume.
  • a concentrated solution of TBE buffer was prepared by diluting to 1 liter, 108 grams of TRIS base, 55 grams of boric acid and 9.3 grams of Disodium EDTA «2H 2 0.
  • the pH of the concentrated TBE buffer should be adjusted to 8.3, if appropriate.
  • 48 grams of ultra-pure grade urea (8 M) was added to the stock solution containing the 30% acrylamide. 10 ml of TBE buffer was added and the urea was dissolved in this solution.
  • 50 ⁇ l (per 100 ml of acrylamide solution) of N,N,N',N'-tetramethylethyl- enediamine(TEMED) and 1 ml (per 100 ml) of 10% ammonium persulfate was added and the solution was mixed well.
  • the gel was poured between two glass plates into the electrophoresis apparatus and the comb was inserted immediately.
  • the gels were then wrapped in a plastic folder placed next to Kodak X-OMAT AR film and exposed for 10 minutes.
  • Figure 6 is an illustrative example of the autoradiograph of the present invention using the method of the present invention. From the autpradio- graph, one can easily determine the presence of the target sequence by the appearance of the 18 mer band derived from the 28 mer oligonucleotide. It can be seen that the use of glycerine in the assay with only one labeled oligonucleotide probe is feasible in the range between 10% to 40% by volume glycerine. A more preferred range is about 40% by volume glycerine.
  • the restriction amplification assay is initiated by combining the HPV 16 substrate, the partially hairpin oligonucleotide labeled probe, Pst I buffer and deionized water. Stock solutions of the reaction components are first prepared. The oligonucleotide is diluted to a 0.5 x 10 "5 molar solution. The HPV 16 stock is diluted with distilled water to form a 100 ⁇ g/ml stock solution. The Pst I restriction enzyme stock solution contains 50 units/ ⁇ l.
  • Each reaction assay contained 1 ⁇ l (100 ng) of HPV 16, 2 ⁇ l of the labeled partial hairpin oligonucleotide, 2 ⁇ l of unlabeled partial hairpin oligonucleotide, 5 ⁇ l of Pst I buffer containing 100 mM NaCl, 10 mM TRIS-HCl at pH 7.7, 10 mM MgCl 2 , 1 mM DTT and 100 ⁇ g/ml BSA. 5 ⁇ l of Pst I is used. Distilled water was added in varying quantities such that the final volume in each assay tube is equal to 50 ⁇ l.
  • the first tube (#1) contains 2 ⁇ l of the partial hairpin oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled partial hairpin oligonucleotide, 5 ⁇ l Pst I buffer, 0 ⁇ l HPV 16 and 36 ⁇ l distilled water.
  • the second tube (#2) contains 2 ⁇ l of the partial hairpin oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled partial hairpin oligonucleotide, 5 ⁇ l Pst I buffer, 1 ⁇ l of a control DNA which does not contain the HPV 16 sequence and 35 ⁇ l distilled water.
  • the third tube (#3) contains 2 ⁇ l of the partial hairpin oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled partial hairpin oligonucleotide, 5 ⁇ l Pst I buffer, 1 ⁇ l of HPV 16 and 35 ⁇ l distilled water.
  • the fourth tube (#4) contains 2 ⁇ l of the partial hairpin oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled partial hairpin oligonucleotide, 5 ⁇ l Pst I buffer, 5 ⁇ l of HPV 16 and 31 ⁇ l distilled water. Pst I is then added to the four assay tubes described above and the tubes are boiled for 5 to 10 minutes and then cooled at room temperature for approximately 10 minutes. After cooling the samples, the samples are placed in an incubator at 37°C. To initiate the assay, 5 ⁇ l of Pst I enzyme is added to each assay tube. The assay is run for 2 hours.
  • a concentrated solution of TBE buffer is prepared by diluting to 1 liter, 108 grams of TRIS base, 55 grams of boric acid and 9.3 grams of Disodium EDTA «2H 2 0.
  • the pH of the concentrated TBE buffer should be adjusted to 8.3, if appropriate.
  • 48 grams of ultra-pure grade urea (8 M) is added to the stock solution containing the 30% acrylamide. 10 ml of TBE buffer is added and the urea is dissolved in this solution .
  • 50 ⁇ l (per 100 ml of acrylamide solution) of N,N,N' ,N'-tetramethylethylene- diamine(TEMED) and 1 ml (per 100 ml) of 10% ammonium persulfate is added and the solution is mixed well.
  • the gel is poured between two glass plates into the electrophoresis apparatus and the comb is inserted im ⁇ mediately.
  • the gels are then wrapped in a plastic folder placed next to Kodak X-OMAT AR film and exposed for 10 minutes.
  • the restriction amplification assay is initiated by combining the HPV 16 substrate, the partial hairpin oligonucleotide labeled probe, Fokl buffer containing 10 mM TRIS-HCl, pH 7.4; 50 mM NaCl; 1 mM EDTA; and 5 mM MgCl 2 and deionized water.
  • Stock solutions of the reaction components are first prepared.
  • the oligonucleotide is diluted to a 0.5 x 10 "5 molar solution.
  • the HPV 16 stock is diluted with distilled water to form a 100 ⁇ g/ml stock solution.
  • the Fokl restriction enzyme stock solution contains 50 units/ ⁇ l.
  • Each reaction assay contained 1 ⁇ l (100 ng) of HPV 16, 2 ⁇ l of the labeled partial hairpin oligonucleotide, 2 ⁇ l of unlabeled partial hairpin oligonucleotide, 5 ⁇ l of Fokl buffer, 5 ⁇ l of Fokl is used. Distilled water was added in varying quantities such that the final volume in each assay tube is equal to 50 ⁇ l. Four assays are run in separate tubes.
  • the first tube (#1) contains 2 ⁇ l of the partial hairpin oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled partial hairpin oligonucleotide, 5 ⁇ l Fokl buffer, 0 ⁇ l HPV 16 and 36 ⁇ l distilled water.
  • the second tube (#2) contains 2 ⁇ l of the partial hairpin oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled partial hairpin oligonucleotide, 5 ⁇ l Fokl buffer, 1 ⁇ l of a control DNA which does not contain the HPV 16 sequence and 35 ⁇ l distilled water.
  • the third tube (#3) contains 2 ⁇ l of the partial hairpin oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled partial hairpin oligonucleotide, 5 ⁇ l Fokl buffer, 1 ⁇ l of HPV 16 and 35 ⁇ l distilled water.
  • the fourth tube (#4) contains 2 ⁇ l of the partial hairpin oligonucleotide labeled with 32 P, 2 ⁇ l of cold unlabeled partial hairpin oligonucleotide, 5 ⁇ l Fokl buffer, 5 ⁇ l of HPV 16 and 31 ⁇ l distilled water.
  • the four assay tubes described above are boiled for 5 to 10 minutes and then cooled at room temperature for approximately 10 minutes.
  • the samples After cooling the samples, the samples are placed in an incubator at 37°C. To initiate the assay, 5 ⁇ l of Fokl enzyme is added to each assay tube. The assay is run for 2 hours.
  • reaction is stopped by adding 10 ⁇ l of a solution containing 80% formamide, 15 mM TRIS-HCl at pH 7.6, 1 mM EDTA, 0.1% w/v bromophenol blue and 0.1% w/v xylene cyanole FF to each assay tube. Each tube is then heated for 5 minutes at 65°C and cooled rapidly.
  • Polyacrylamide gels are prepared by making a stock solution containing 30% acrylamide (19:1 acrylamide:bis) , 19 grams of acrylamide, 1 gram of N,N'-methylenebisacrylamide and enough deionized water to dilute the solution to 67 ml total volume.
  • a concentrated solution of TBE buffer is prepared by diluting to 1 liter, 108 grams of TRIS base, 55 grams of boric acid and 9.3 grams of Disodium EDTA «2H 2 0. The pH of the concentrated TBE buffer should be adjusted to 8.3, if appropriate.
  • 48 grams of ultra-pure grade urea (8 M) is added to the stock solution containing the 30% acrylamide. 10 ml of TBE buffer is added and the urea is dissolved in this solution.

Abstract

La présente invention se rapporte à un procédé, un réactif et un kit destinés à la détermination de la présence de séquences nucléotidiques cibles par amplification par enzyme de restriction. Selon le procédé servant à détecter des séquences d'acide nucléique, une molécule cible contenant un site de restriction spécifique est hybridée avec une sonde marquée contenant une séquence homologue à au moins 28 bases de la molécule cible. La sonde est segmentée par une enzyme de restriction qui libère la sonde afin que celle-ci soit détectée si elle s'hybride avec la cible spécifique. Ainsi, la sonde segmentée se régénère constamment et est éminemment détectable si la séquence cible est présente dans le dosage.
PCT/US1992/009606 1991-11-12 1992-11-12 Dosage par amplification par enzyme de restriction WO1993010264A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP5509342A JPH07505760A (ja) 1991-11-12 1992-11-12 制限増幅検定法
EP92924356A EP0620861A4 (fr) 1991-11-12 1992-11-12 Dosage par amplification par enzyme de restriction.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US79017491A 1991-11-12 1991-11-12
US790,174 1991-11-12

Publications (1)

Publication Number Publication Date
WO1993010264A1 true WO1993010264A1 (fr) 1993-05-27

Family

ID=25149854

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/009606 WO1993010264A1 (fr) 1991-11-12 1992-11-12 Dosage par amplification par enzyme de restriction

Country Status (4)

Country Link
EP (1) EP0620861A4 (fr)
JP (1) JPH07505760A (fr)
CA (1) CA2122734A1 (fr)
WO (1) WO1993010264A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997012062A1 (fr) * 1995-09-29 1997-04-03 Lynx Therapeutics, Inc. Detection de polynucleotide par amplification en isothermie
KR20000025029A (ko) * 1998-10-07 2000-05-06 박한오 제한효소 절편의 배열화된 증폭의 전개방법

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4935357A (en) * 1986-02-05 1990-06-19 New England Biolabs, Inc. Universal restriction endonuclease
US5102784A (en) * 1990-05-04 1992-04-07 Oncor, Inc. Restriction amplification assay

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FEBS Letters, Vol. 212, No. 2, issued February 1987, KRUPP et al., "Simplified In Vitro Synthesis of Mutated RNA Molecules", pages 271-275; see page 273, Figure 2. *
See also references of EP0620861A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997012062A1 (fr) * 1995-09-29 1997-04-03 Lynx Therapeutics, Inc. Detection de polynucleotide par amplification en isothermie
US5747255A (en) * 1995-09-29 1998-05-05 Lynx Therapeutics, Inc. Polynucleotide detection by isothermal amplification using cleavable oligonucleotides
KR20000025029A (ko) * 1998-10-07 2000-05-06 박한오 제한효소 절편의 배열화된 증폭의 전개방법

Also Published As

Publication number Publication date
CA2122734A1 (fr) 1993-05-27
JPH07505760A (ja) 1995-06-29
EP0620861A4 (fr) 1995-07-19
EP0620861A1 (fr) 1994-10-26

Similar Documents

Publication Publication Date Title
US5102784A (en) Restriction amplification assay
EP0167238B1 (fr) Procédé d'analyse des polynucléotides par déplacement et réactif des complexes polynucléotidiques à cet effet
US5093245A (en) Labeling by simultaneous ligation and restriction
US5830643A (en) Partially double-stranded oligonucleotide and method for forming oligonucleotide
US6072043A (en) Optimally fluorescent oligonucleotides
JP3415150B2 (ja) 分枝核酸プローブの使用方法
EP1062367B1 (fr) Methodes de detection d'acide nucleique utilisant des zymogenes et des kits associees
CA1340775C (fr) Methode pour la detection de sequences specifiques d'acides nucleiques
US6090592A (en) Method for performing amplification of nucleic acid on supports
EP0357011B1 (fr) Amplification et détection des séquences d'acides nucléiques
US6013439A (en) Detection of differences in nucleic acids
US20070059690A1 (en) "Met/fret based method of target nucleic acid detection whereby the donor/acceptor moieties are on complementary strands"
CA1254114A (fr) Epreuve de deplacement de polynucleotides utilisant une proteine de recombinaison et une trousse de diagnostic
EP0379369A2 (fr) Amplification d'acides nucléiques utilisant une amorce
JPH0437720B2 (fr)
CA2196604A1 (fr) Appareil et procede pour realiser une amplification d'acides nucleiques sur des supports
EP0912767A2 (fr) Procedes pour detecter et amplifier des sequences d'acide nucleique au moyen d'oligonucleotides modifies ayant une temperature de fusion specifique de la cible accrue
Iovannisci et al. Ligation amplification and fluorescence detection of Mycobacterium tuberculosis DNA
WO2008109945A1 (fr) Analyse d'acide ribonucléique
US5451502A (en) Restriction amplification assay
Gillespie The magic and challenge of DNA probes as diagnostic reagents
US7070925B1 (en) Method for determining the presence of an RNA analyte in a sample using a modified oligonucleotide probe
WO1993010264A1 (fr) Dosage par amplification par enzyme de restriction
US6492120B1 (en) Nucleic acid hybridization assay utilizing tricyclic target and signal amplification
WO2000043543A1 (fr) Detection des differences entre polynucleotides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2122734

Country of ref document: CA

Ref document number: 1992924356

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1992924356

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1992924356

Country of ref document: EP