IE921940A1 - Polynucleotide determination with selectable cleavage sites - Google Patents
Polynucleotide determination with selectable cleavage sitesInfo
- Publication number
- IE921940A1 IE921940A1 IE194092A IE921940A IE921940A1 IE 921940 A1 IE921940 A1 IE 921940A1 IE 194092 A IE194092 A IE 194092A IE 921940 A IE921940 A IE 921940A IE 921940 A1 IE921940 A1 IE 921940A1
- Authority
- IE
- Ireland
- Prior art keywords
- polynucleotide
- support
- reagent
- nucleic acid
- label
- Prior art date
Links
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6823—Release of bound markers
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
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- C07F9/24—Esteramides
- C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
- C07F9/2429—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of arylalkanols
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- 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
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Abstract
Novel methods for assaying a nucleic acid analyte are provided, which employ polynucleotides having oligonucleotide sequences substantially homologous to a sequence of interest in the analyte, where the presence or absence of hybridization at a predetermined stringency provides for the release of a label from a support. Particularly, various techniques are employed for binding a label to a support, whereupon periodate cleavage of a linkage between the label and support results in release of label, which can be detected as indicative of the presence of a particular oligonucleotide sequence in a sample. The method finds use in diagnosis of disease, genetic monitoring, and analysis of nucleic acid mixtures.
Description
PQLYMtfCLEOIIPB PETEBttltfATIPN WITH
SELECTABLE CLEAVAGE SITES
Psscrlfttipn
Technical Field
The invention relates generally to the incorporation of selactebly cleavable sites into oligonucleotide chains, and nor· particularly relates to novel reagents useful for introducing perlodate-deevable linkages into oligonucleotide chains. The invention also relates to nethods of using the novel reagents in biochemical assays.
Background
The ability to synthesize oligonucleotide sequences at will and to clone polynucleotide sequences prepared by synthetic procedures or obtained froa naturally occurring sources has greatly expended the opportunities for detecting the presence of specific sequences in an extended oligonucleotide sequence, e.g., chromosome(s), mixture of sequences, mRNAs, or the like, interest in specific sequences may involve the diagnosis of the presence of pathogens, the determination of the presence of alleles, the presence of lesions in a host genome, the detection of a particular mRNA or the monitoring of a modification of a cellular host, to mention only a fev illustrative opportunities. While the use of antibodies to perform assays diagnostic of the presence of various antigens in samples has seen an explosive expansion in techniques and protocols since the advent of radioimmunoassay, there has been until recently
-2no parallel activity in th· area of the DNA probes. Therefore, for the most part, detection of sequences has involved various hybridisation techniques requiring the binding of a polynucleotide sequence to a support and employing a radiolabeled probe.
In view of the increasing capability to produce oligonucleotide sequences in large amounts in an economical way, the attention ot investigators vill be directed to providing for simple, accurate and efficient techniques for detecting specific oligonucleotides sequences. Desirably, these techniques will be rapid, minimize the opportunity for technician error, be capable of automation, and allow for simple and accurate methods of detection. Toward this end, there have already been efforts to provide for means to label oligonucleotide probes vith labels other than radioisotopes and for improving the accuracy of transfer of DNA sequences to a support from a gel, as well as improved methods for derivatizing oligonucleotides to allow for binding to a label. There continues to be a need for providing new protocols which allow for flexibility in detecting DNA sequences of interest in a variety of situations where the DNA may come from diverse sources.
Overview of the Art
Meinkoth and Wahl, Anal. Biochemistry (1984)
138:267-284, provide an excellent review of hybridization techniques. Leary, et al., Proc. Natl. Acad. Sci. PSA (1983) ££:4045-4049, describe the use of biotinylated DNA in conjunction vith an avidin-enzyme oonjugate for detection of specific oligonucleotide sequences. Rank! et el., Gene (1983) 21:77-65 describe what they refer to as a sandwich hybridization for detection of oligonucleotide sequences. Pfsuffer and Relmrich, J. of
Biol. Chem. (1975) £££:867-876 describe the coupling of
-jgu*nosine-5'-0-(3-thiotriphosphate) to Sepharose 4B. Bauman, at al., J\_Qf Hlstocham. and Cvtochem^ (1981) 22:227-237, describe the 3'-labeling of RNA with fluoreseers. PCT Application W0/8302277 desoribes tha addition to ONA fragments of modified ribonucleotides for labeling and methods for analysing such DNA fragments. Rans and Xurs, Nuol. Acids Res. (1984) 12:3435-3444, describe tha covalent linking of enzymes to oligonucleotides. Wallace, ONA Recombinant Technology 10 (Woo, S., Ed.) CRC Press, Boca Raton, Florida, provides a general background of the use of probes in diagnosis.
Chou and Herigan, N, Ena. J. of Wed. (1983) 308:921-925. describe the use of a radioisotope labeled probe for the detection of CMV. Inman, Methods in Enzymol. 34B, 24 (1974) 30-59, describes procedures for linking to polyacrylamides, while Parikh, et al., Methods_in Enzymol. 34B, 24 (1974) 77-102, describe coupling reactions vith agarose. Alwine, et al., Proc, Natl.
Acad, Sci. USA (1977) 2£·’5350-5354, describe a method of transferring oligonucleotides from gels to a solid support for hybridisation. Chu, et al., Nucl. Acids Res. (1983) ll;6513-6529, describe a technique for derlvetlsing terminal nucleotides. Ho, et al.. Biochemistry (1981) 20:64-67. describe dsrivatlzing terminal nucleotides through phosphate to form esters. Ashley and MacDonald, Anal. Biochem. (1984) 140:95-103, report a method for preparing probes from a eurface bound template. These references which describe techniques are incorporated herein by reference in support of the preparation of labeled oligonucleotides.
Disclosure of the Invention
Methods are provided for the detection of specific nucleotide sequences employing e solid support, at least one label, and hybridisation involving a sample
-4and a labeled probe, where the presence or absence of duplex formation results in the ability to Modify the spatial relationship between the support and label(s). Exemplary of the technique is to provide a cleavage site between the label and support through duplexing of a labeled probe and sample DNA, where the duplex is bound to a support. The cleavage site may then be cleaved resulting in separation of the support and the label (s). Detection of the presence or absence of the label may then proceed in accordance with conventional techniques.
A primary advantage of the invention over the art is that the present method enables one to distinguish between specific and nonspecific binding of the label. That is, in the prior art, label is typically detected on a solid support, i.e., the sample is affixed to the support and contacted with a complementary, labeled probe; duplex formation is then assayed on the support. The problem with this method is that label can and does bind to the support in the absence of analyte. This direct binding of the label to the support is referred to herein as nonspecific binding. If any significant amount of nonspecific binding occurs, label will be detected on the support regardless of the presence of analyte, giving false positive results.
By contrast, in the present method, label is detected only when the analyte of interest is present,
i.e., only specific binding is detected. Zn a preferred embodiment, this is done by introducing a cleavage site between a support and the selected label, through a duplex between the sample and one or more probes. The cleavage site may ba a restriction endonuclease cleavable site, as described in U.S. Patent No, 4,775,619 (cited and incorporated by reference above), or it may be one of a number of types of chemically cleavable sites, e.g., as described in D.s.
5Application Serial No. 07/251,152, th· parent application hereto.
The present application ie directed to a new class of chemically cleavable sites. These cleavable sites ere extremely stable vith respect tc the conditions and reagents used in hybridization assays, but are readily cleavabls, when cleavage is desired, with periodate ion. The present invention is also directed to polynucleotide reagents containing the cleavable sitae, and to reagents useful in polynucleotide synthesis, i.e., monomeric reagent· vhich also contain the cleavable eitee and vhich may be readily Incorporated into a polynucleotide chain. These various reagents are readily synthesized in high yield and, like the cleavable sites themselves, are quits stable under a variety of conditions.
Brief Description of the Drawings
Figure 1 illustrates the difference between specific and nonspecific binding of a label to a solid support.
Figures 2A through 2D schematically illustrate the preferred method of the invention, wherein a selectively cleavable site is introduced between a support and a label through an analyts/probe complex.
Modes for Carrying Out the Invention
Before describing the methods end reagents of the invention in detail, it is to be understood that this invention is not limited to the particular protocols or materials described herein as such may, of oourse, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to bs limiting.
-6It must be noted that, as used in this specification and th* appended claims, the singular forms •a, an and the include plural referents unless the content clearly dictate* otherwise. Thus, for example, reference to a cleavage site* includes two or more cleavage sites, reference to a label* includes two or more labels, and the like.
In describing and claiming the present invention, the following terminology will be used in accordance with th* definition* set out below.
Alkyl refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Lower alkyl refer* to an alkyl group of one to eight, more preferably one to six, carbon atoms, and thus includes, for example, methyl, ethyl, propyl, etc.
Alkenyl refers to a branched or unbranched unsaturated hydrocarbon group of 2 to 24 carbon atoms and one or more unsaturated carbon-carbon bonds, such as for example, ethenyl, l-propenyl, 2-propenyl, 1-butenyl, 2isobutenyl, octenyl, decenyl, tetradecenyl, d·'11beptadecadienyl, hexadecenyl, eicosenyl, tetracosenyl and the like. Lower alkenyl refers to an alkenyl group of two to eight, more preferably two to six, carbon atoms, and thus includes, for example, ethenyl, 1-propenyl, 2-propenyl, l-butenyl, 2-isobutenyl and octenyl.
Alkylene refers to a difunctional saturated branched or unbranched hydrocarbon chain containing from l to 6 carbon atoms, and includes, for example, methylene (-CH2-), ethylene (-CH2-CH2-), propylene (-CH2-CH2-CH2-), 2-methylpropylen* [-CH2-CH(CH3) -CH2-J, hexylene end the like.
-7Alkenylene refer· to a difunctional, branched or unbranched unsaturated hydrocarbon group ef 2 to 34 carbon atom· and one or more uneaturated carbon-carbon bonds, such as, for example, l,3-propyl-l-ene, l,4-but-25 enylene, l,5-pent-2-enylene, end i,6-hex-3-enylene.
Aryl· refers to a phenyl or 1- or 2-naphthyl group. Optionally, these groups ere substituted vith one to three, sore preferably one to tvo, lover alkyl, lover alkoxy, hydroxy, amino, nitro and/or mercapto substituents.
Arylalkylene refers to an aryl group as is defined herein vhich is attached to one end of an alkylene group as is defined herein.
Cycloalkyl refers to a saturated hydrocarbon 15 ring group having from 3 to β carbon atoms, and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclohexyl, methylcydohexyl, cyclooctyl, and the like.
Cycloalkylene refers to a saturated 20 hydrocarbon containing a cycloalkyl group as is defined herein attached to one end of an alkylene group as is defined herein. The term includes, for example, cyclohexyl methylene, cyclopropyl methylene, cyclobutyl ethylene, 6-cyclooctyl hexylene, and the like.
Cyclooxyalkylene refers to a cycloalkylene group as defined herein which contains one or more ether oxygen atoms.
The present invention involves the detection of specific sequences using hybridization, vhereby duplexing of the sample DNA and a probe affects the ability to modify the spatial relationship betveen s label and a support. In this manner, the presence or absence of a particular sequence in a sample can be readily determined by the amount of label vhich is freed into the medium.
Th· subject method allows for varying protocols and reagents where the sample nucleic acid may be bound to a support or free in solution. In a preferred , embodiment, the method involves forming a nucleic acid 5 duplex where a label is separated from a support by a cleavable bond, so that the amount of label released under conditions providing selective cleavage is a measure of the presence and amount of a sequence of Interest in a nucleic acid sample. The selectable cleavage site may be as a result of formation of a restriction enzyme recognition site through homoduplexing, or the presence of such selectable cleavage site in the single-stranded polynucleotide chain may be a result of the prior introduction of such site into the single-stranded chain.
A reagent will be employed which will include a polynucleotide sequence having an oligonucleotide sequence of Interest that hybridizes to the nucleic acid analyte. This reagent vill sometimes be referred to herein as a capture probe, which in the present method, is bound to the selected solid support. A labeling probe vill also be employed, which may or may not include the sequence of interest.
in the first, preferred embodiment, the subject method involves the forming of e polynucleotide duplex in a hybridization medium resulting in a label bound to a support through a selectable cleavage site. Various protocols may be employed where the sample DNA is bound to a support or dispersed in a solution.
In order to distinguish the various nucleotide sequences involved, the following terms will be used:
nucleic acid sample - sample suspected of containing a nucleic acid sequence having an oligonucleotide sequence of interest;
nucleic acid analyte - DNA or RNA in aaid nucleic said sample having an oligonucleotide sequence of interest;
oligonucleotide sequence of interest *- a DNA or 5 RNA sequence vhich aay be all or part of a nucleotide chain, usually at least six bases, acre usually at least about 10 bases, preferably at least about 16 bases, which say be 5Kb or more, usually not sore than .2Jtb, vhich is diagnostic of a property to be detected, where the property nay be a gene or sequence diagnostic of a hereditary trait, pathogen, etc.;
polynucleotide sequence - DNA or RNA sequences employed as rsagents for detection of the oligonucleotide sequence of interest, vhich polynucleotide sequence nay be labeled or unlabeled, bound or unbound to a support, and nay or nay not include a sequence complementary to the oligonucleotide sequence of interest. There will be one to tvo polynucleotide sequences, vhich individually or in conjunction vith the nucleic acid analyte vill act as a bridge between a label end a support, vith a selectably cleavable site intermediate the label end support; and selectably cleavable site - a functionality or plurality of functionalities vhich can be selectively cleaved vith periodate.
For convenience of description, the preferred embodiment of the subject invention wherein a selectable clehvage site is created vill be divided into four primary sub-embodiments. Zn the first of these (see Fig.
2A) the reagent employed is a single component, vhich is a polynucleotide joined proximal to one end to a support end joined proximal to the opposite end to one or more detectable labels. The polynucleotide includes e cleavable site intermediate the support end label.
-10In the second cess (See Fig. 2B), the reagent employed will have two components which will wary with whether the nucleic ecid sample is bound or unbound to a , support. Where the nucleic acid eanple is bound to the 5 support, the two components will be (l) a bridging polynucleotide sequence and (2} a polynucleotide sequence complementary and hybridising to a portion of the bridging polynucleotide sequence. The complementary polynucleotide sequence is labeled. Besides having a sequence duplexing with the complementary sequence, the bridging polynucleotide sequence will have a region duplexing with the oligonucleotide sequence of interest.
Where the sample nucleic acid is in solution, the two components will be (1} a first polynucleotide sequence bound to a support, which has a region complementary to a sequence present in the nucleic sold analyte, which sequence may or may not define the oligonucleotide sequence of interest; and (2) a labeled second polynucleotide sequence which as a region complementary to a sequence present in the nucleic acid analyte, which region is subject to the same limitations as the region of the first polynucleotide sequence. At least one of the duplexed regions will define a sequence of interest. Either the first or second polynucleotide sequence contains the selectable cleavage site.
In a third case (see Fig. 2C), the analyte ia bound to a support and the reagent employed is a single component which is a labeled polynucleotide sequence having a region complementary to the oligonucleotide sequence of interest and containing the selectable cleavage site.
In a fourth case (see Fig. 2D), a capture probe is provided which is a polynucleotide chain bound to a solid support via a linkage Υ, and at its opposing end is complementary to a first sequence present in the
nucleic sold analyte. A labeling probe comprising a labeled second polynucleotide chain has a region complementary to a second sequence in the analyte that ie distinct from and does not overlap vith the first sequence. The linkage designated Y in Fig. 2D represents any conventional means of binding e probe to a support. The linkage X* represents the periodatecleavable linkage.
The selectable cleavage sites vhich are the 10 focal point of the present invention are all periodatecleavable linkages having the structural formula -Rj-O-X0-R2-, vherein R2 end R2 are Independently selected from the group consisting of alkylene, alkenylene, cycloalkylene, cycloalkenylene, cyclooxyalkylene, aryl, aralkylene, and combinations thereof, where these terms are as defined above, and X ie the periodate-cleavable linkage itself. Examples of periodate-cleavable moieties vhich WXM may represent include:
w \? \/ 0H 0H. O OH , ΝΗίΓοΗ
0 and NHR NHR where R is hydrogen or alkyl (typically lover alkyl).
To carry out the present method, the nucleic acid containing sample vill be combined with the 35 appropriate reagent under conditions where duplex
12formation occurs between complementary sequences. The mixture is allowed to hybridize under conditions ef predetermined stringency to allow for at least hetsroduplex formation or homoduplex formation over an oligonucleotide sequence of interest. After s sufficient time for hybridization to occur, the support may be separate from the supernatant and washed free of at least substantially all of the non-specif ically bound label.
The oligonucleotides bound to the support ere then treated vith periodate, vhich results In cleavage of at least one strand and release of label bound to support.
Depending upon the presence of a particular sequence in the sample resulting in duplex formation, release of the label(s) bound to the support will be observed. Various protocols may bs employed, where normally the supernatant medium may be assayed for the presence of the label, although in some instances the support may be measured. Protocols and reagents may be employed, where a physical separation of the support from the supernatant may or may not be required.
The subject method can be used for the detection of oligonucleotide sequences, either DNA or RNA, in a vide variety of situations. One important area of interest is the detection of pathogens, viruses, bacteria, fungi, protozoa, or the like, vhich can infect a particular host. See for example, U.S. Patent No. 4,358,535. Another area of interest is the detection of alleles, mutations or lesions present in the genome of a host, such as involved in amniocentesis, genetic counseling, host sensitivity or susceptibility determinations, and monitoring of cell populations. A third area of interest is the determination of the presence of RNA for such diverse reasons as monitoring transcription, detecting RNA viruses, differentiating organisms through unexpressed RNA, and the like. Other ,E 921940
area* of interest, which are intended to he illustrative, hut not totally inclusive, include monitoring modified organisms for th* presence of extrachroaosomal DNA or . integrated DNA, amplifications of DNA sequences,- the 5 maintenance of such sequences.
The physiological samples may he obtained from a vide variety of sources as is evident from the varied purposes for vhich the subject method may be used.
Sources aay include various physiological fluids, such a* excreta, e.g., stool, sputum, urine, saliva, etc.;
plasma, blood, serum, ocular lens fluids, spinal fluid, lymph, and the like. The sample may be used without modification or may be modified by expanding the sample, cloning, or th* like, to provide an isolate, so that there is an overall enhancement of the DNA or RNA and reduction of extraneous RNA or DNA. Viruses may be plated on a lawn of compatible cells, so as to enhance the amount of viral DNA; clinical isolates may be obtained by the sample being streaked or spotted on a nutrient agar medium and individual colonies assayed; or the entire sample introduced into a liquid broth and the cells selectively or non-selsctivsly expanded. The particular manner in vhich the sample is treated vill be dependent upon the nature of the sample, the nature cf the DNA or RNA source, the amount of oligonucleotide sequence of interest vhich is anticipated as being present as compared to the total amount of nucleic acid present, a* veil as the sensitivity of the protocol and label being employed.
Either the sample nucleic acid or the reagent polynucleotide may be bound, either covalently or non-covalently, but in any event non-diffueively, to the support. (In the case of the embodiment represented by Fig. 2D, th* capture probe alone is bound to the solid support.) Where a sample nucleic acid is bound to the
support, various supports have found particular use and to the extant, those supports vill be preferred. These supports include nitrocellulose filters, diazotised paper, scteola paper, or other support which provides such desired properties as low or no non-specific binding, retention of the nucleic add sample, ease of manipulation, and allowing for various treatments, such as growth or organisms, washing, heating, transfer, and label detection, a* appropriate.
To the extent that a component of the polynucleotide reagent is bound to the support, the type of support may be greatly varied over the type of support involved vith th* sample oligonucleotide. The support may include particles, paper, plastic sheets, container holder walls, dividers, millipore filters, etc., where the materials may include organic polymers, both naturally occurring and synthetic, such as polysaccharides, polystyrene, polyacrylic add and derivatives thereof, e.g., polyacrylamide, glass, ceramic, metal, carbon, polyvinyl chloride, protein, and the like. The various materials may be functionalized or non-functionalized, depending upon whether covalent or non-covalent bonding is desired.
Where th* sample nucleic acid is bound to the support, depending upon the particular support, heating may be sufficient for satisfactory binding of the nucleic add. In other situations, diazo groups may be employed for linking to the nucleic acid. Where, however, the polynucleotide reagent component is bound to the support, a vide variety of different techniques may be employed for ensuring the maintenance of th* polynucleotide reagent bound to the support. For example, supports can be functionalized, to have active amino groups for binding, resulting from the binding of alkylamines, hydrazides, or thioeemicarbazides to the support. One
-15can than add, by mean* of a terminal transferase, a ribonucleotide to a DNA polynucleotide reagent. Upon glycol cleavage vith an appropriate oxidant, e.g·, , periodate, osmium tetroxide plus hydrogen peroxide, lead 5 tetraacetate, or the like, a dialdehyde is formed, vhich vill then bind to the amino group on tha surface to provide a monosubstituted amino or disubstituted amino group. Alternatively, one can provide for a maleimide group vhich vith thiophosphate vill form the alkylthioester. Various techniques described by Parikh, et al., supra and by Inman, supra for agarose and polyacrylamide may be employed, vhich techniques may have application vith other materials.
The total number of polynucleotide reagent components on the support available in the assay medium vill vary, for the most part being determined empirically. Desirably, a relatively high concentration per unit surface area of polynucleotide to available functional groups on the support should be employed, so long as the polynucleotide density does not interfere vith hybridization.
The size of the polynucleotide vill vary videly, usually being not less than about 15 bases and may be 50 bases or more, usually not exceeding about 500 bases, more usually not exceeding 250 bases. There vill usually be a region in the polynucleotide reagent component homologous vith a sequence in the nucleic acid sample, usually the sequence of interest, of at least six bases, usually at least 12 bases. The region for hybridization may be 15 bases or more, usually not exceeding about Ikbp, where perfect homology 1* not required, it being sufficient that there be homology to at least about 50*, more preferably homology to et least 80*. (By percent homology is intended complementary.
ignoring non-coaple»«ntary insertions which may loop out, insertions being greater than five baaes.)
Particularly, where one is interested in a group of allelic genes, a number of different strains, or related species, where the messenger RNA or genomic portion is highly conserved but nevertheless is subject to polymorphisms, it vill frequently be desirable to prepare a probe which reflects the differences and optimizes the homology for all the sequences of interest, iu as agaxnsb any particular seguawcs.
The label of the labeled polynucleotide reagent component may be joined to the polynucleotide sequence through the selectively cleavable eite or through e link which is retained during the essay. A vide variety of labels may be employed, where the label »&y provide for a detectable signal or means for obtaining e detectable signal.
Labels therefore include such diverse substituents as ligands, radioisotopes, enzymes, fluorescers, chemiluminsscers, enzyme suicide inhibitors, enzyme cofactors, enzyme substrates, or other substituent vhlch can provide, either directly or indirectly, a detectable signal.
where ligands are involved, there will normally be employed a receptor which specifically binds to the ligand, e.g., biotin and avidin, 2,4'-dinitrobenzene and anti(2,4-dinitrobenzene)IgG, etc., where the receptor will be substituted vith appropriate labels, as described above. In this manner, one can augment the number of labels providing for a detectable signal per polynucleotide sequence.
For the most part, the labels employed for use in immunoassays can be employed in the subject essays. These labels are illustrated in U.S. Patent Nos.
3,850,752 (enzyme); 3,853,914 (spin label); 4,180,016 —17— (fluorescer); 4,174,384 (f luorescer and quencher) ; 4,160,645 (catalyst); 4,277,437 (chemilualnescer); 4,318,707 (quenching particle); and 4,318,890 (enzyme substrate).
illustrative fluorescent and chemiluminescent labels include fluorescein, rhodamlne, dansyl, umbelliferone, biliprotelns, luminol, «to.
Illustrative enzymes of interest include horse radish peroxidase, glucose-6-phosphat* dehydrogenase, acetylcholinesterase, £-galactoaidaee, e-amylase, uricase, malate dehydrogenase, etc. That is, the enzymes of interest vill primarily be hydrolases and oxidoreductases.
The manner in which the label becomes bound to the polynucleotide sequence vill vary widely, depending upon the nature of the label. As already indicated, a ribonucleotide may be added to the oligonucleotide sequence, cleaved, and th* resulting dialdthyde conjugated to an amino or hydrazine group. The permanence of the binding may be further enhanced by employing reducing conditions, which results in th* formation of an alkyl amine. Alternatively, the label may be substituted with an active halogen, such as alpha-bromo or -chloroacetyl. This may be linked to a thiophosphate group or thiopurine to form a thioether. Alternatively, the label may have maleimide functionality, where a mercapto group present on the polynucleotide vill form a thioether. The terminal phosphate of the polynucleotide may be activated with carbodiinide, where the resulting phosphorimidazolide vill react vith amino groups or alcohols to result in phosphoramidates or phosphate esters. Polypeptide bonds may be formed to amino modified purines. Thus, one has a vide latitude in the choice of label, th* manner of linking, and th* choice of linking group.
-1βBy combining the polynucleotide reagent with the sample, any nucleic acid analyte present vill become bound to the support. The amount of label released from . the support upon cleavage of the selectable cleavage site 5 will be related to the presence of analyte, where the amount of analyte may also be determined quantitatively.
The modification of the spatial relationship between the label and the support can be achieved in a number of ways. As indicated, there can be at least one recognition site common to the probe and the same polynucleotide, thus releasing the probe from the support.
Ligand-substituted nucleotides oan be employed where the ligand does not give a detectable signal directly, but bonds to a receptor to which is conjugated one or more labels. Illustrative examples include biotinylated nucleotides which will bind to avidin, haptens which will bind to immunoglobulins, and various naturally occurring compounds which bind to proteinaceous receptors, such as sugars with lectins, hormones end growth factors with cell surface membrane proteins, and the like.
In the embodiment represented by Fig. 2D, the selectable cleavage site may be introduced in one of two ways.
First, a crosslinking compound may be incorporated into the capture probe 1 itself, i.e., at position X as indicated in the figure. Any number of crosslinking agents may be used for this purpose, the only limitation being that the cleavage site introduced into the capture probe must be cleavable with periodate. Examples of suitable crosalinking reagents for introducing periodate-cleavable linkages are bis** carboxylate with a sulfur-sulfur bridge (available from
Pierce Chemicals) and disuccinimidyl tartarata (DST).
The selectable cleavage site may also be . Introduced by appropriate modification of the capture probe prior to attachment to the solid support. This method involves preparation of e polynucleotide.having the structure
where X is or contains the periodate-cleavable linkage as described above, where DNAX is a first strand of DNA,
DNA3 is a second strand of DNA, end Rx and Rj are as defined earlier. In e particularly preferred embodiment,
-Rj-O-X-O-Rj- is
OH OH
This compound may then be attached to a solid support, using conventional means well knovn in the art, to give the capture probe illustrated in Fig. 2D. Such e compound is prepared using a reagent vherein the 1,2-diol system is protected as the dibenzoyl compound during DNA synthesis end vhich further contain* an acid-sensitive, base-stable protecting group (such as dimethoxytrityl, or DMT) at substituent Yx end hydrogen or a phosphorus derivative such ee phosphoramidite, phosphotriester, phosphodiester, phosphite, K-pho*phonate, or phosphorothioate at substituent Y2:
Yx-O-Rx-O-X-O-R2-O-Y2
allowing for incorporation into a DNA fragment using standard phosphoramidite chemistry protocols. An exemplary compound may be represented by
OH OH
OCH, wherein *DMTM represents dimethoxytrityl end iPr represents isopropyl.
As in the embodiment represented by rigs.
2A-2C, the embodiment of Fig. 20 enables detection of specifically bound label in solution (and thus accurate measurement of analyte 2) while nonspecifically bound label 6 remains bound to the solid support 5.
A vide variety of support* and technique* for non-diffusiv* binding of oligonucleotide chains have been reported in th* literature. For a review, see Meinkoth and Wahl, Anal. Biochem. (1984) 124?267-284. Support* include nitrocellulose filters, vhere temperatures of
80*C for 2 hr suffices, diazotized papers, where bonding occurs without further activation, ecteola paper, etc. Agarose beads can be activated with cyanogen bromide for direct reaction vith DNA. (Bauman, et al., J. Hlstochem. Cvtochem, (1981) 22:227-237); or reacted vith cyanogen bromide and a diamine followed by reaction vith an _-haloacetyl, e.g., bromoacetyl or vith an active carboxylic substituted olefin, e.g., maleic anhydride, to provide beads capable of reacting vith a thiol functionality present on a polynucleotide chain. For example, DNA can he modified to form a _-thiophosphate for coupling. (Ffeuffer and Hilmreich, J. Biol. Chem. (1975) 250:867-878.1 It is also possible to synthesize by chemical means an oligonucleotide bound to a Teflon support and then fully deblock th* materiel without removing it (Lohrmann, et al., DNA (1984) 2.:122).
21—
In view of the vid· diversity of label· and reagents, the common aspects of the method vill be described, followed by a fev exemplary protocol*. Common to the procedures vill be hybridization. The hybridization can be performed at varying degrees of stringency, so that greater or lesser homology is required for duplexing. For the most part, aqueous media vill be employed, vhich may have a mixture of various other components. Particularly, organic polar solvents may be employed to enhance stringency. Illustrative solvents include dimethylformamide, dimethylacetamide, dimethylsulfoxide, that is, organic solvents vhich at the amounts employed, are miscible vith water. Stringency can also be enhanced by increasing salt concentration, so that one obtains an enhanced ionic strength. Also, increasing temperature can be used to stringency. In each case, the reverse direction results in reduced stringency. Other additives may also be used to modify the stringency, such as detergents.
The period of time for hybridization will vary vith the concentration of the sequence of interest, the stringency, the length of the complementary sequences, and the like. Usually, hybridization vill require at least about 15 min, and generally not more than about 72 hr, more usually not more than about 24 hr. Furthermore, one can provide for hybridization at one stringency and than wash at a higher stringency, so that heteroduplexes lacking sufficient homology are removed.
The nucleic acid sample vill be treated in a variety of ways, where one may employ the intact genome, mechanically sheared or restriction enzyme digested fragments of the genome, varying from about .5kb to 30kb, or fragments vhich have been segregated according to size, for example, by electrophoresis. In some instances, the sequences of interest vill be cloned
22seguences, vhich have been cloned in an appropriate vector, for example, a single-stranded DNA or ANA virus, e.g., 1(13.
; Included in the assay medium may bs other additives including buffers, detergents, s.g., 8DS,
FJLcoll, polyvinyl pyrrolidons and foreign DNA, to minimize non-specific binding. All of these additives find illustration in the literature, and do not need to be described in detail here.
In accordance vith a particular protocol, the sample nucleic acid end polynucleotide reagent(e) are brought together in the hybridization medium at the predetermined stringency. After a sufficient time for hybridization, the support vill bs washed at least ones vith s medium of greater or lesser stringency than the hybridization medium. The support vith the bound polynucleotide and analyte vill then be contacted vith the necessary reactants (includes physical treatment, e.g., light) for cleaving the selectable cleavage site, providing for single- or double-stranded cleavage. For the most part hydrolase enzymes vill bs used, such as restriction endonucleases, phosphodiesterases, pyrophosphatase, peptidases, esterases, etc., although other reagents, such as reductants, Sllman's reagent, or light may find use. After cleavage, the support and the supernatant may or may not be separated, depending upon the label and the manner of measurement, and the amount of label released from the support determined.
To further illustrate the subject invention, s few exemplary protocols vill be described. In the first exemplary protocol, a microtiter plate is employed, where fluorescent labeled polynucleotides are bound to the bottom of each wall. DNA from a pathogen which has been cloned, le restricted vith one or more restriction snzymee to provide fragments of from about 0.5 2kb. The
-23fragments ar· isolated under wild basic conditions for denaturing and dispersed in the hybridisation medium, vhich is then added sequentially to the various veils, e each of the veils having different sequences which are 5 specifically homologous vith sequences of different •trains of a particular pathogen species.
The veils ere saintalned at an elevated temperature, e.g., 50*C, for sufficient time for hybridization to occur, vhereupon the supernatant is removed and valla are thoroughly washed repeatedly with a buffered medium of lover stringency than the hybridization medium. Duplex formation results in a recognition site for a restriction enzyme common to all of the strains. To each wall is then added a restriction enzyme medium for digestion of double-stranded DNAs vhich are digested result in the release of the fluorescent label into the supernatant. The supernatant is aspirated from each of the wells and irradiated. The amount of fluorescence is then determined as indicative of the presence of the sequence of interest, in this manner, one can rapidly screen for which of the strains is present, by observing the presence of fluorescence in the liquid phase.
In the second exemplary protocol, one employs a column containing glass beads to vhich are bound unlabeled polynucleotide. To the column is then added the sample nucleic acid containing DNA fragments obtained from mammalian cells. The fragments range from about 0.5 to lOkb. The sample DNA is dispersed in an appropriate hybridization medium and added to the column and retained in the column for sufficient time for hybridisation to occur. After the hybridization of the sample, the hybridization medium is released from the column and polynucleotide reagent labeled vith horse radish peroxidase (HRP) through a disulfide linkage is added in
a second hybridization medium under more stringent conditions than the first medium end the second medium released in the column for sufficient time for hybridization to occur. The labeled polynucleotide has a sequence complementary to the sequence of Interest. The hybridization medium is evacuated froa the column.
The column may then be washed one or more times vith a medium of higher stringency to remove any polynucleotide sequences vhich have insufficient homology vith the labeled polynucleotide. Ellman's reagent is then added to the column resulting in cleavage of the disulfide linkage and release of the HRP. The HRP containing medium is evacuated froa the column and collected, as well ae a subsequent wash to ensure that freed enzyme ie not held up in the column. The resulting medium vhich contains the HRP label may now be assayed for the KRP label. Instead of HRP a vide variety of other enzymes can bs used vhich produce products vhich can be detected spectrophotometrically or fluorometrically.
In a third protocol, the nucleic acid sample ie non-diffusively bound to one end of a nitrocellulose filter by absorbing the sample vith the filter and heating at 80*c for 2 hr. The filter is washed and then added under hybridization conditions to e hybridization solution of e polynucleotide labeled with umbelliferone through an ester linkage to an alkylcarboxy substituted adenine. The labeled polynucleotide has a sequence complementary to the sequence of interest. After sufficient time for hybridization the filter le removed from the hybridization medium, washed to remove non-specifically bound nucleotides, and than submerged in e measured solution of en esterase. The rate of increase of fluorescence is monitored as a measure of the amount of analyte in the nucleic acid sample.
25In another protocol, dipstick can be used of a plastic material where a holder is employed which holds a strip having a labeled polynucleotide sequenced complementary to the analyte sequence vith a polyfluoresceinylated terminus. The nucleio acid sample is prepared in th* appropriate hybridization medium and the dipstick introduced and hybridization allowed to proceed. After sufficient time for th* hybridization to have occurred, the dipstick is removed and washed to remove any non-specific binding polynucleotide. The presence of a polynucleotide sequence ef interest results in the formation of a restriction enzyme recognition site and the dipstick is then introduced into the restriction enzyme reaction mixture and digestion allowed to proceed.
After sufficient time for digestion to have proceeded, the dipstick is removed, thoroughly washed, and the fluorescence in the solution read, while fluorescence above a baseline value indicates the presence of the analyte.
In another protocol, the polynucleotide reagent components are a first polynucleotide which has a sequence complementary to one region of the nucleio acid analyte and is bound to the walls of wells of a microtiter plate and a labeled second polynucleotide which ha* a sequence complementary to another region of the nucleic acid analyte. The label is the result of tailing the polynucleotide vith Ne-aninohexyl deoxy adenosine triphosphate umbel liferyl carboxamide.
The nucleic acid sample is introduced into the wells vith an excess of th* labeled polynucleotide under hybridizing conditions. After sufficient time for hybridization, the hybridization solution is aspirated out of the wells, the wells washed and the residual DNA in the wells depurinated by adding a solution of 90% formic acid and w w
-26heating at 60*C for 1 hr or adding piperidine and heating at 90“C for 30 min.
Alternatively, the label can ba a result of ligating the polynucleotide to be labeled with-an excess of an oligomer obtained by treating poly-dA with chloroacetaldehyde according to Silver and Feisht, Biochemistry (1962) 21:6066 to produce the fluorescent Ke-ethenoadenosi»e. Release of the label is achieved with micrococcal nuclease in a solution of lOOugM CaCl^ io for 1 hr at 37 ®c.
In both instances the fluorescence of the polymer is substantially diminished due to self-quenching. Upon dissolution, a substantial enhancement in fluorescence is observed. Thus, non-specifically bound labeled polynucleotide resistant to the depolymerization would not interfere with the observed signal. Furthermore, one could measure the rate of increase of fluorescence as a quantitative measure of nucleic acid analyte, since the background fluorescent level would be low. Instead of self quenching, systems can be employed where fluorescers and quenchers alternate or in two component reagent systems, non-quenching fluorescers are present on one component and quenchers are present on the other component.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMBttM,
Synthesis of a PCL (Periodate-Cleavable Linker):
In this experiment, the abbreviation X represents D,L-l,4-bis- (4-(2-hydroxyethyl)phenoxy)-2,3butanedlol, the abbreviation X'“ represents 2,3isopropylidene, and the abbreviation MDMT-X(Bza)-BCE represents 2-(4-(4(4-(2-dimethoxytrityloxy)
ethyl) phenoxy-2,3-di (benzoyloxy) -butane-oxy]phenyl) ethyl2-cyanoethy 1-N, N-di isopropyl phosphor en id ite. DMTX(Bz2)-BCE2 has the structural formula
DMT-O-tCHjJj-^-O-yy-O-^-iCH,),—O-R^
OHOH OCHj
To a mixture of 4-hydroxyphenylethenol (21.4 g;
155 mmole) end l,4-dibromo-2,3-butane diol (19.3 g; 78 mmole) dissolved in 400 ml of absolute ethanol was added NaOH (26 ml of a 6H solution in water). The reaction mixture was gently refluxed for 18 hours. After cooling to room temperature, half of the solvent was removed in vacuo (-200 ml) and this solution added dropwise to 1000 ml vater vith rapid stirring. The formed precipitate vas filtered off and dried extensively in a vacuum desiccator over solid NaOH (10 g) to assist drying to give 11.9 g (33 mmole) X (yield 42%).
Compound X (33 mmole) was dissolved in THF, the solution (330 ml) then filtered, and N,Ndimethylaminopyridine (100 mg) and triethylamine (27 ml J 200 mmole) were added, and finally t-butyldimethylsilyl chloride (TBDHS-Cl) (19.8 g; 132 mmole) vas added to the above mixture. After 18 hours at 20*C, all starting material had been consumed (as verified by tic analysis) and methanol (50 ml) vas added to consume excess TBDMS-C1 (25 min). The reaction mixture vas concentrated to a small volume, diluted vith ethyl acetate (250 ml) end vaahed vith 1 x 250 ml 5% NaHCO3 end 1 x 250 ml 80% sat. ag. NaCl solution. After drying tha organio phase over solid Na2SO4, the solvent was removed in vacuo. Tha crude TBDMSj-X was dissolved in pyridine, cooled to 0*C, and benzoyl chloride (132 mmole), dissolved in 125 ml
CH2C12, was added dropwise. The reaction mixture was allowed to warm to room temperature and left for 18 hours. The pyridine solvent was removed in vacuo and the residue dissolved in ethyl acetate. After en aqueous work-up as described above, the crude TBDMS2XBz2 (30 mmole) was dissolved in 200 ml THF containing 100 ml cone, acetic acid, and tetrabutylammonium fluoride (100 ml IM in THF) was added, and the reaction mixture left at 4*C for 18h. Host of the solvent was then removed in vacuo and the residue in ethyl acetate was treated with solid NaHCOj to neutralize excess acetic acid, washed and dried as described above to give X(Bz2) (30 mmole; 17.0 g). This material was used vithout purification and treated in pyridine vith 30 mmole DMT-C1. After 18 hours, the solvent was removed in vacuo, the residue in ethyl acetate was washed and dried, as described above, to give 27 g crude DMT-X(Bt2). The crude product was purified on a large column of silica using CH2C12/1% triethylamine as solvent system to give pure DMT-X(Bz2) (13.3 g; 15 mmole). This purified material was converted to the 2-cyanoethyl phosphoramidite· as follows: DMTX(Bz2) (15 mmole) was dissolved in 50 ml CH2C12 containing Ν,Ν-diisopropylethylamlne (13.1 ml; 75 mmole) and cooled to O’C. To this solution under argon was added vith a syringe 2-cyanoethoxy-N,N-diisopropylaminochlorophosphine (3.3 ml; 15 mmole). After -30 min, the reaction was complete, and after diluting vith 500 ml ethyl acetate the organic phase was washed vith 2 x 500 ml 5% NaHCOj and 2 x 500 ml 80% sat. NaCl. After drying over solid Na2SO4, the solution was filtered end evaporated to dryness to give 18 g white foam of DMT— X(Bz2)BCE amidite. Tha crude amidite was purified on a column of silica gel eluted vith CH2Cl2/ethyl acetate/triethylamine (45:45:10 v/v) to give a white foam
of pure DMT-X(Bz2)BCS amidite (14.2 9; 13 Wool·). (NMR 31P f 144 ppm; coupling efficiency 97*.)
Conversion of the 1,2-diol linkage te other t periodate-cleavable species as disclosed herein nay be readily effected using conventional techniques well-known in the art of synthetic organic chemistry.
Synthesis of X'»
Compound X (25 g, 6» mmole) vas suspended in
CHjCN (200 ml) and 50 ml 2,2-dlmethoxypropana was added. Anhydrous tosic acid (10 ml of a o.lM solution in acetonitrile) vas added during the next few hours. A mostly clear solution resulted. After 19 hours the solution vas filtered and 10 ml H2O was added and left for 10 min to destroy excess reagent and other byproducts. Pyridine (50 ml) vas added and the reaction mixture va* concentrated in vacuo to give 25 g of X' product. Without purification, this material vas converted to the DMT and further to the BCB phosphoramidite as described for X(Bz2) above.
Results:
Th* fully protected DMT-X(Bz2)BCE amidite wa* incorporated into an oligomer, 5'-T10-X-Tls-3', on a solid support. The fragment vas deprotected vith dichloroacetic acid and ammonium hydroxide first at 20*c for l hour (to cleave the succinate linkage), then at 60*C to remove the benzoyl groups on the X moiety. No cleavage of the oligomer va* observed. A sample of th* test oligomer in vater vas treated with 100 mM Naio4 in water at 4‘C for one hour. Excess reagent was then reduced vith ribose. Polyacrylamide gel electrophoresis (PAGE) showed the cleavage to be complete, giving rise to two shorter fragments, 5*-T10 x-3* and 5'-x-T16-3', where
X-O-(CH2)2-CeB4-O-CH2-CHO. PAGE resultst
Lane 1: Tlfi
2: 3: 5'-T10-X-Tls-3', NH4OH 20*C/I hr 4: Same as lene 3 but NR40H <0-*c/ie hr 5: Same as lane 4 after exposure to Malo* for 1 hr 6: Same as lane 5
It is evident from the above results that the subject method provides for a simple, rapid and accurate approach for detecting specific polynucleotide sequence· from diverse sources. The method provides for high sensitivity and great flexibility in allowing for different types of labels which involve detectable signals which have been employed in Immunoassays. Thus, the subject method can be readily adapted to use in conventional equipment for immunoassays which are capable of detecting radioactivity, light adsorption in spectrophotometers and light emission in fluorometers or scintillation counters. The subject method is applicable to any DNA sequence and can use relatively small probes to reduce false positive and minimize undesirable heteroduplexing. By cleavage of the label from the support for measurements, background values can be greatly reduced, since the reading can occur away from the support. Also, there is e further redirection in background due to the necessity to cleave the label from the polynucleotide chain. The subject method can therefore provide for the accurate and economical determination of DNA sequences for diagnosing disease, monitoring hybrid DNA manipulations, determining genetic traits, and ths like.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will
be obvious that certain changes and modification» may be practiced within the scope of the appended claims.
Claims (17)
1. λ method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid 5 analyte present in a nucleic acid sample, said method comprising: combining under hybridising conditions said nucleic acid sample vith a polynucleotide reagent, wherein one of said sample or a component of said reagent 10 is bound to a support and hybridization of said analyte and said polynucleotide reagent results in a label being bound to said support through a selectable cleavage site -Rj-O-X-O-Rj- , wherein Rj and R a are independently selected from the group consisting of alkylene, 15 alkenylene, cycloalkylene, eycloalkenylene, cyclooxyalkylene, eryl, aralkylane, and combinations thereof, and X is a periodats-cleavable linkage; substantially freeing said support of label bound to said support other than through said selectable 20 cleavage site; cleaving said cleavage site vith a periodate reagent; and detecting label free of said support. 25
2. The method of claia 1, wherein said polynucleotide reagent comprises a first polynucleotide capture probe bound to a support and a second polynucleotide label probe, wherein said first and second probes have oligonucleotide sequences complementary to 30 sequences present in said analyte so as to form duplexes therevith under said hybridising conditions, at least ons of said oligonucleotide sequence* being a sequence of interest, wherein eaid capture probe contain* said selectable cleavage site.
3. A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in s nucleic acid sample, said method comprisingz a 5 combining under hybridising conditions in an aqueous medium, said nucleic acid sample vith e polynucleotide reagent, where one of said sample or a component of said reagent is bound to a support and hybridisation of said analyte and said polynucleotide re10 agent results in s label being bound to eaid support through a selectable cleavage sits -Rj-0-X-0-R a -, wherein R x and R a are independently selected from the group consisting of alkylene, alkenylene, cycloalkylene, cycloalkenylene, cyclooxyalkylene, aryl, aralkylene, and 15 combinations thereof, and X is a periodata-cleavabla linkage; separating said support having bound polynucleotide reagent and nucleic acid analyte from said aqueous medium; 20 washing said support with a medium of different hybridising stringency from said aqueous medium to remove label bound to said support other than through said selectable cleavage site; cleaving said cleavage site vith e periodate 25 reagent; and detecting label free of said support.
4. The method of claim 3, wherein said polynucleotide reagent comprises a first polynucleotide 30 capture probe bound to a support and a second polynucleotide label probe, wherein said first and second probes have oligonucleotide sequences complementary to ssquancss present in said analyte to fora duplexes therewith under said hybridising conditions, at least one 35 of said oligonucleotide sequences being a sequence of interest, wherein said capture prob· contains said •electable cleavage site.
5. The method of claim 1, wherein Xi* 5 selected from the group consisting of wherein R is hydrogen or alkyl.
6. selected from The method of claim 3, wherein X is the group consisting of NHR OH and NHR NHR wherein R is hydrogen or alkyl.
7. λ probe useful for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sampl·, comprising a polynucleotide sequence bound proximal to one end to e 5 support end at is opposite end having a sequence complementary to said sequence of interest, said probe containing in addition a selectable cleavage site -R x -OX-O-R 2 ~, vherein Rj and R a are independently selected from the group consisting of alkylene, alkenylene, 10 cycloalkylene, cycloalkenylene, cyclooxyalkylene, aryl, eraIkylene, and combinations thereof, and X is a pariodate-cleavable linkage.
8. λ polynucleotide reagent having the 15 structure 0 0 I I 5· -HO 5 ’ (DNAJ] 3 ' -O-P-O-R^O-X-O-Rj-O-P-O- 5 ’ [DNAj J 3 '-OH where DNA^ is a first strand of DNA, DNA^ is a second strand of DNA, Rj and R a are Independently selected from the group consisting of alkylene, alkenylene, 25 cycloalkylene, cycloalkenylene, cyclooxyalkylene, aryl, aralkylene, and combinations thereof, end X ie a periodate-cleavable linkage.
9. Th· polynucleotide reagent of claim a, wherein X is selected from the group consisting of wherein R is hydrogen or alkyl.
10. The polynucleotide reagent of claim 6, 20 wherein -Rj-O-X-O-Rj- is (CH 2 ) 2 25 OHOH
11. λ reagent useful in polynucleotide synthesis, given by the structure Yj-O-Rj-O-X-O-Rj-O-Yj wherein Rj and R 2 are independently selected fro* the group consisting of alkylene, alkenylene, oycloalkylene, 35 cycloalkenylene, cyclooxyalkylene, aryl, aralkylene, and combinations thereof, X is a periodate-oleavable linkage, Yj is an acid-sensitive, base-stable protecting group, and Y 2 is selected from the group consisting of hydrogen, phosphoranidite, phoaphotrtester, phosphodiester, « < 5 phosphite, H-phosphonate and phosphorothioats.
12. The reagent of claim 11, having the structural formula OH OH 0°¼ 15 wherein DMT represents dimethoxytrityl and lPr represents isopropyl. -3813. A method according to claim 1 or 3 for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, substantially as hereinbefore described with reference to the accompanying drawings.
13. 14. A method according to claim 1 or 3 for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample, substantially as hereinbefore described and exemplified.
14. 15. A probe according to claim 7, substantially as hereinbefore described and exemplified.
15. 16. A polynucleotide reagent having the structure given and defined in claim 8, substantially as hereinbefore described and exemplified.
16.
17. A reagent having the structure given and defined in claim 11, substantially as hereinbefore described and exemplified.
Applications Claiming Priority (1)
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US71667591A | 1991-06-17 | 1991-06-17 |
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IE194092A IE921940A1 (en) | 1991-06-17 | 1992-07-01 | Polynucleotide determination with selectable cleavage sites |
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JP (3) | JPH06509707A (en) |
KR (1) | KR940701454A (en) |
CA (1) | CA2110591A1 (en) |
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DE19856796A1 (en) * | 1998-12-09 | 2000-06-15 | Biochip Technologies Gmbh | Cleavage of chemically synthesized oligo- / polynucleotides at a predetermined location |
US6642000B1 (en) * | 1999-11-12 | 2003-11-04 | University Of Chicago | PCR amplification on microarrays of gel immobilized oligonucleotides |
US7122314B2 (en) | 2002-01-30 | 2006-10-17 | Id Biomedical Corporation | Methods for detecting vancomycin-resistant microorganisms and compositions therefor |
CN103733047B (en) | 2011-08-11 | 2016-03-30 | 奥林巴斯株式会社 | The detection method of intended particle |
JP6010034B2 (en) | 2011-08-30 | 2016-10-19 | オリンパス株式会社 | Target particle detection method |
JP5940644B2 (en) | 2012-02-22 | 2016-06-29 | オリンパス株式会社 | Target particle detection method |
WO2013140890A1 (en) | 2012-03-21 | 2013-09-26 | オリンパス株式会社 | Method for detecting target nucleic acid molecule |
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US4775619A (en) * | 1984-10-16 | 1988-10-04 | Chiron Corporation | Polynucleotide determination with selectable cleavage sites |
US5118605A (en) * | 1984-10-16 | 1992-06-02 | Chiron Corporation | Polynucleotide determination with selectable cleavage sites |
CA1339351C (en) * | 1987-10-15 | 1997-08-26 | Michael S. Urdea | Nucleic acid multimers and amplified nucleic acid hybridization assays using same |
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1992
- 1992-06-12 CA CA002110591A patent/CA2110591A1/en not_active Abandoned
- 1992-06-12 JP JP5501087A patent/JPH06509707A/en active Pending
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JP2002369699A (en) | 2002-12-24 |
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WO1992022671A1 (en) | 1992-12-23 |
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