EP0610215A1 - Dosage de polynucleotides a l'aide de sites de clivage pouvant etre selectionnes - Google Patents
Dosage de polynucleotides a l'aide de sites de clivage pouvant etre selectionnesInfo
- Publication number
- EP0610215A1 EP0610215A1 EP92914302A EP92914302A EP0610215A1 EP 0610215 A1 EP0610215 A1 EP 0610215A1 EP 92914302 A EP92914302 A EP 92914302A EP 92914302 A EP92914302 A EP 92914302A EP 0610215 A1 EP0610215 A1 EP 0610215A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- polynucleotide
- support
- reagent
- bound
- label
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- 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
- C07F9/22—Amides of acids of phosphorus
- 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
-
- 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
Definitions
- the invention relates generally to the incorporation of selectably cleavable sites into oligonucleotide chains, and more particularly relates to novel reagents useful for introducing periodate-cleavable linkages into oligonucleotide chains.
- the invention also relates to methods of using the novel reagents in biochemical assays.
- oligonucleotide sequences The ability to synthesize oligonucleotide sequences at will and to clone polynucleotide sequences prepared by synthetic procedures or obtained from naturally occurring sources has greatly expanded 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 few illustrative opportunities.
- Methods are provided for the detection of specific nucleotide sequences employing a solid support, at least one label, and hybridization involving a sample and 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.
- 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.
- label is detected only when the analyte of interest is present, i.e., only "specific" binding is detected.
- 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 be 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 U.S. Application Serial No. 07/251,152, the parent application hereto.
- the present application is directed to a new class of chemically cleavable sites. These cleavable sites are extremely stable with respect to the conditions and reagents used in hybridization assays, but are readily cleavable, when cleavage is desired, with periodate ion.
- the present invention is also directed to polynucleotide reagents containing the cleavable sites, and to reagents useful in polynucleotide synthesis, i.e., monomeric reagents which also contain the cleavable sites and which may be readily incorporated into a polynucleotide chain. These various reagents are readily synthesized in high yield and, like the cleavable sites themselves, are quite stable under a variety of conditions.
- Figure 1 illustrates the difference between specific and nonspecific binding of a label to a solid support.
- FIGS. 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 analyte/probe complex.
- 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” refers 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, 1-propenyl, 2-propenyl, 1-butenyl, 2- isobutenyl, octenyl, decenyl, tetradecenyl, ⁇ 8 ' 11 - heptadecadienyl, 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, 1-butenyl, 2-isobutenyl and octenyl.
- Alkylene refers to a difunctional saturated branched or unbranched hydrocarbon chain containing from 1 to 6 carbon atoms, and includes, for example, methylene (-CH 2 -) , ethylene (-CH 2 -CH 2 -) , propylene (-CH 2 -CH 2 -CH 2 -) , 2-methylpropylene [-CH 2 -CH(CH 3 )-CH 2 -], hexylene t-(CH 2 ) 6 -] and the like.
- Alkenylene refers to a difunctional, branched or unbranched unsaturated hydrocarbon group of 2 to 24 carbon atoms and one or more unsaturated carbon-carbon bonds, such as, for example, 1,3-propyl-l-ene, l,4-but-2- enylene, l,5-pent-2-enylene, and l,6-hex-3-enylene.
- Aryl refers to a phenyl or 1- or 2-naphthyl group. Optionally, these groups are substituted with one to three, more preferably one to two, lower alkyl, lower alkoxy, hydroxy, amino, nitro and/or mercapto substituents.
- Arylalkylene refers to an aryl group as is defined herein which is attached to one end of an alkylene group as is defined herein.
- Cycloalkyl refers to a saturated hydrocarbon ring group having from 3 to 8 carbon atoms, and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclohexyl, methylcyclohexyl, cyclooctyl, and the like.
- Cycloalkylene refers to a saturated 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, whereby duplexing of the sample DNA and a probe affects the ability to modify the spatial relationship between a 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 which is freed into the medium.
- the subject method allows for varying protocols and reagents where the sample nucleic acid may be bound to a support or free in solution.
- the method involves forming a nucleic acid 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 will sometimes be referred to herein as a "capture probe", which in the present method, is bound to the selected solid support.
- a labeling probe will also be employed, which may or may not include the sequence of interest.
- the subject method involves the forming of a 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.
- nucleic acid sample - sample suspected of containing a nucleic acid sequence having an oligonucleotide sequence of interest nucleic acid analyte - DNA or RNA in said nucleic said sample having an oligonucleotide sequence of interest
- nucleic acid analyte There will be one to two polynucleotide sequences, which individually or in conjunction with the nucleic acid analyte will act as a bridge between a label and a support, with a selectably cleavable site intermediate the label and support; and selectably cleavable site - a functionality or plurality of functionalities which can be selectively cleaved with periodate.
- the preferred embodiment of the subject invention wherein a selectable cleavage site is created will be divided into four primary sub-embodiments.
- the reagent employed is a single component, which is a polynucleotide joined proximal to one end to a support and joined proximal to the opposite end to one or more detectable labels.
- the polynucleotide includes a cleavable site intermediate the support and label.
- the reagent employed will have two components which will vary with whether the nucleic acid sample is bound or unbound to a support.
- the two components will be (1) a bridging polynucleotide sequence and (2) a polynucleotide sequence complementary and hybridizing to a portion of the bridging polynucleotide sequence.
- the complementary polynucleotide sequence is labeled.
- the bridging polynucleotide sequence will have a region duplexing with the oligonucleotide sequence of interest.
- 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 acid 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.
- the analyte is 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.
- a capture probe which is a polynucleotide chain bound to a solid support via a linkage "Y", and at its opposing end is complementary to a first sequence present in the nucleic acid analyte.
- a labeling probe comprising a labeled second polynucleotide chain has a region complementary to a second sequence in the analyte that is distinct from and does not overlap with the first sequence.
- the linkage designated "Y” in Fig. 2D represents any conventional means of binding a probe to a support.
- the linkage "X" represents the periodate- cleavable linkage.
- the selectable cleavage sites which are the focal point of the present invention are all periodate- cleavable linkages having the structural formula -R ⁇ -0-X- 0-R 2 -, wherein R ⁇ and R 2 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 is the periodate-cleavable linkage itself.
- Examples of periodate-cleavable moieties which "X" may represent include:
- the nucleic acid containing sample will be combined with the appropriate reagent under conditions where duplex formation occurs between complementary sequences.
- the mixture is allowed to hybridize under conditions of predetermined stringency to allow for at least heteroduplex formation or homoduplex formation over an oligonucleotide sequence of interest.
- the support may be separate from the supernatant and washed free of at least substantially all of the non- ⁇ pecifically bound label.
- the oligonucleotides bound to the support are then treated with periodate, which results in cleavage of at least one strand and release of label bound to support.
- the subject method can be used for the detection of oligonucleotide sequences, either DNA or RNA, in a wide variety of situations.
- One important area of interest is the detection of pathogens, viruses, bacteria, fungi, protozoa, or the like, which 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 areas of interest which are intended to be illustrative, but not totally inclusive, include monitoring modified organisms for the presence of extrachromosomal DNA or integrated DNA, amplifications of DNA sequences, the maintenance of such sequences
- the physiological samples may be obtained from a wide variety of sources as is evident from the varied purposes for which the subject method may be used.
- Sources may include various physiological fluids, such as 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 the 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-selectively expanded.
- the particular manner in which the sample is treated will be dependent upon the nature of the sample, the nature of the DNA or RNA source, the amount of oligonucleotide sequence of interest which is anticipated as being present as compared to the total amount of nucleic acid present, as well 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-diffusively, to the support.
- the capture probe alone is bound to the solid support.
- various supports have found particular use and to the extent, those supports will be preferred. These supports include nitrocellulose filters, diazotized paper, ecteola paper, or other support which provides such desired properties as low or no non-specific binding, retention of the nucleic acid sample, ease of manipulation, and allowing for various treatments, such as growth or organisms, washing, heating, transfer, and label detection, as appropriate.
- the type of support may be greatly varied over the type of support involved with the 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 polysaccharide ⁇ , polystyrene, polyacrylic acid 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.
- sample nucleic acid is bound to the support, depending upon the particular support, heating may be sufficient for satisfactory binding of the nucleic acid.
- diazo groups may be employed for linking to the nucleic acid.
- the polynucleotide reagent component is bound to the support, a wide variety of different techniques may be employed for ensuring the maintenance of the polynucleotide reagent bound to the support.
- supports can be functionalized, to have active amino groups for binding, resulting from the binding of alkylamines, hydrazides, or thiose icarbazides to the support.
- a terminal transferase a ribonucleotide to a DNA polynucleotide reagent.
- an appropriate oxidant e.g., periodate, osmium tetroxide plus hydrogen peroxide, lead tetraacetate, or the like
- a dialdehyde is formed, which will then bind to the amino group on the surface to provide a monosubstituted amino or disubstituted amino group.
- a maleimide group which with thiophosphate will form the alkylthioester.
- Various techniques described by Parikh, et al., supra and by Inman, supra for agarose and polyacrylamide may be employed, which techniques may have application with other materials.
- the total number of polynucleotide reagent components on the support available in the assay medium will 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 with hybridization.
- the size of the polynucleotide will vary widely, 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 will • usually be a region in the polynucleotide reagent component homologous with 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 16 bases or more, usually not exceeding about lkbp, where perfect homology is not required, it being sufficient that there be homology to at least about 50%, more preferably homology to at least 80%. (By percent homology is intended complementary, ignoring non-complementary insertions which may loop out, insertions being greater than five bases.)
- the label of the labeled, polynucleotide reagent component may be joined to the polynucleotide sequence through the selectively cleavable site or through a link which is retained during the assay.
- a wide variety of labels may be employed, where the label may provide for a detectable signal or means for obtaining a detectable signal.
- Labels therefore include such diverse substituents as ligands, radioisotopes, enzymes, fluorescers, chemilu inescers, enzyme suicide inhibitors, enzyme cofactors, enzyme substrates, or other substituent which can provide, either directly or indirectly, a detectable signal.
- ligands 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 with appropriate labels, as described above. In this manner, one can augment the number of labels providing for a detectable signal per polynucleotide sequence.
- a receptor which specifically binds to the ligand, e.g., biotin and avidin, 2,4'-dinitrobenzene and anti(2,4-dinitrobenzene)IgG, etc.
- the labels employed for use in immunoas ⁇ ays can be employed in the subject assays. These labels are illustrated in U.S. Patent Nos. 3,850,752 (enzyme); 3,853,914 (spin label) ; 4,160,016 (fluorescer) ; 4,174,384 (fluorescer and quencher); 4,160,645 (catalyst); 4,277,437 (chemiluminescer) ; 4,318,707 (quenching particle); and 4,318,890 (enzyme substrate) .
- Illustrative fluorescent and chemiluminescent labels include fluorescein, rhodamine, dansyl, umbelliferone, biliproteins, luminol, etc.
- Illustrative enzymes of interest include horse radish peroxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, ⁇ -galactosidase, ⁇ -amylase, uricase, malate dehydrogenase, etc. That is, the enzymes of interest will primarily be hydrola ⁇ es and oxidoreductases.
- the manner in which the label becomes bound to the polynucleotide sequence will vary widely, depending upon the nature of the label.
- a ribonucleotide may be added to the oligonucleotide sequence, cleaved, and the resulting dialdehyde conjugated to an amino or hydrazine group.
- the permanence of the binding may be further enhanced by employing reducing condition ⁇ , which re ⁇ ult ⁇ in the formation of an alkyl amine.
- 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.
- the label may have maleimide functionality, where a ercapto group present on the polynucleotide will form a thioether.
- the terminal phosphate of the polynucleotide may be activated with carbodiimide, where the resulting phosphorimidazolide will react with amino group ⁇ or alcohols to result in phosphoramidate ⁇ or phosphate e ⁇ ter ⁇ .
- Polypeptide bond ⁇ may be formed to amino modified purine ⁇ .
- any nucleic acid analyte present will become bound to the support.
- the amount of label released from the support upon cleavage of the selectable cleavage site will be related to the presence of analyte, where the amount of analyte may also be determined quantitatively.
- the modification of the ⁇ patial 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 relea ⁇ ing the probe from the ⁇ upport.
- Ligand- ⁇ ubstituted nucleotides can 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 and growth factors with cell surface membrane proteins, and the like.
- the selectable cleavage site may be introduced in one of two ways.
- 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.
- suitable crosslinking reagents for introducing periodate-cleavable linkages are bis- carboxylate with a sulfur-sulfur bridge (available from Pierce Chemicals) and disuccinimidyl tartarate (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 a polynucleotide having the structure
- X is or contains the periodate-cleavable linkage as described above, where DNA ⁇ is a first strand of DNA, DNA 2 is a second strand of DNA, and R ⁇ and R are as defined earlier.
- -R ⁇ -0-X-0-R 2 - is
- This compound may then be attached to a solid support, using conventional means well known in the art, to give the capture probe illustrated in Fig. 2D.
- a compound is prepared using a reagent wherein the 1,2-diol system is protected as the dibenzoyl compound during DNA synthesis and which further contains an acid-sensitive, base-stable protecting group (such as dimethoxytrityl, or "DMT") at substituent ⁇ and hydrogen or a phosphorus derivative such as phosphoramidite, phosphotriester, phosphodiester, phosphite, H-phosphonate, or phosphorothioate at substituent Y 2 :
- an acid-sensitive, base-stable protecting group such as dimethoxytrityl, or "DMT”
- a phosphorus derivative such as phosphoramidite, phosphotriester, phosphodiester, phosphite, H-phosphonate, or phosphorothioate at substituent Y 2 :
- An exemplary compound may be represented by
- Fig. 2D 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.
- supports and techniques for non-diffusive binding of oligonucleotide chains have been reported in the literature. For a review, see Meinkoth and Wahl, Anal. Biochsm. (1984) 138:267-284. Supports include nitrocellulose filters, where 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 with DNA.
- Illustrative solvents include di ethylformamide, dimethylacetamide, dimethylsulfoxide, that is, organic solvents which at the amounts employed, are miscible with water.
- Stringency can also be enhanced by increasing salt concentration, so that one obtains an enhanced ionic strength.
- 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 with the concentration of the seguence of interest, the stringency, the length of the complementary sequences, and the like. Usually, hybridization will 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 then wash at a higher stringency, so that heter ⁇ duplexes lacking sufficient homology are removed.
- the nucleic acid sample will 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 which have been segregated according to size, for example, by electrophoresis.
- the sequences of interest will be cloned sequences, which have been cloned in an appropriate vector, for example, a single-stranded DNA or RNA virus, e.g., M13.
- the assay medium may be other additives including buffers, detergents, e.g., SDS, Ficoll, polyvinyl pyrrolidone 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.
- the sample nucleic acid and polynucleotide reagent(s) are brought together in the hybridization medium at the predetermined stringency. After a sufficient time for hybridization, the support will be washed at least once with a medium of greater or lesser stringency than the hybridization medium.
- the support with the bound polynucleotide and analyte will then be contacted with the necessary reactants (includes physical treatment, e.g., light) for cleaving the selectable cleavage site, providing for single- or double-stranded cleavage.
- the necessary reactants include physical treatment, e.g., light
- hydrolase enzymes will be used, such as restriction endonucleases, phosphodiesterases, pyrophosphatase, peptidases, esterases, etc., although other reagents, such as reductants, Ellman's reagent, or light may find use.
- 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.
- a few exemplary protocols will be described.
- a microtiter plate is employed, where fluorescent labeled polynucleotides are bound to the bottom of each well.
- DNA from a pathogen which has been cloned is restricted with one or more restriction enzymes to provide fragments of from about 0.5 2kb.
- the fragments are isolated under mild basic conditions for denaturing and dispersed in the hybridization medium, which is then added sequentially to the various wells, each of the wells having different sequences which are specifically homologous with sequences of different strains of a particular pathogen species.
- the wells are maintained at an elevated temperature, e.g., 60°C, for sufficient time for hybridization to occur, whereupon the supernatant is removed and wells are thoroughly washed ⁇ repeatedly with a buffered medium of lower stringency than the hybridization medium. Duplex formation results in a recognition site for a restriction enzyme common to all of the strains. To each well is then added a restriction enzyme medium for digestion of double-stranded DNAs which 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.
- an elevated temperature e.g. 60°C
- the second exemplary protocol one employs a column containing glass beads to which 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 hybridization to occur.
- the hybridization medium is released from the column and polynucleotide reagent labeled with horse radish peroxidase (HRP) through a disulfide linkage is added in a second hybridization medium under more stringent conditions than the first medium and 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 from the column.
- the column may then be washed one or more times with a medium of higher stringency to remove any polynucleotide sequences which have insufficient homology with 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 from the column and collected, as well as a subsequent wash to ensure that freed enzyme is not held up in the column.
- the resulting medium which contains the HRP label may now be assayed for the HRP label.
- HRP a wide variety of other enzymes can be used which produce products which can be detected spectrophotometrically or fluorometrically.
- the nucleic acid sample is non-diffusively bound to one end of a nitrocellulose filter by absorbing the sample with the filter and heating at 80°C for 2 hr.
- the filter is washed and then added under hybridization conditions to a hybridization solution of a 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.
- the filter is removed from the hybridization medium, washed to remove non-specifically bound nucleotides, and then submerged in a measured solution of an esterase.
- 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 with a polyfluoresceinylated terminus.
- the nucleic acid sample is prepared in the appropriate hybridization medium and the dipstick introduced and hybridization allowed to proceed. After sufficient time for the hybridization to have occurred, the dipstick is removed and washed to remove any non-specific binding polynucleotide.
- a polynucleotide sequence of 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.
- the polynucleotide reagent components are a first polynucleotide which has a sequence complementary to one region of the nucleic acid analyte and is bound to the walls of wells of a microtiter plate and a labeled second polynucleotide which has a sequence complementary to another region of the nucleic acid analyte.
- the label is the result of
- the nucleic acid sample is introduced into the wells with an excess of the 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 heating at 60°C for 1 hr or adding piperidine and heating at 90°C for 30 min.
- the label can be 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 (1982) 2L:6066 to produce the fluorescent N -ethenoadenosine. Release of the label is achieved with micrococcal nuclease in a solution of lOOugM CaCl_ for 1 hr at 37°C.
- the reaction mixture was concentrated to a small volume, diluted with ethyl acetate (250 ml) and washed with 1 x 250 ml 5% NaHC0 3 and 1 x 250 ml 80% sat. aq. NaCl solution. After drying the organic phase over solid Na 2 S0 4 , the solvent was removed in vacuo.
- the crude TBDMS 2 -X was dissolved in pyridine, cooled to 0 ⁇ C, and benzoyl chloride (132 mmole), dissolved in 125 ml CH 2 C1 2 , 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.
- the crude TBDMS 2 XBz 2 (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.
- Most of the solvent was then removed in vacuo and the residue in ethyl acetate was treated with solid NaHC0 3 to neutralize excess acetic acid, washed and dried as described above to give X(Bz 2 ) (30 mmole; 17.0 g) .
- the fully protected DMT-X(Bz 2 )BCE amidite was incorporated into an oligomer, 5'-T 10 -X-T 15 -3', on a solid support.
- the fragment was deprotected with dichloroacetic acid and ammonium hydroxide first at 20°C for 1 hour (to cleave the ⁇ uccinate linkage) , then at 60°C to remove the benzoyl groups on the X moiety. No cleavage of the oligomer was observed.
- a sample of the test oligomer in water was treated with 100 mM NaI0 4 in water at 4°C for one hour. Excess reagent was then reduced with ribose.
- the subject method provides for a simple, rapid and accurate approach for detecting specific polynucleotide sequences from diverse sources.
- the method provides for high sensitivity and great flexibility in allowing for differ ⁇ ent types of labels which involve detectable signals which have been employed in immunoassays.
- the subject method can be readily adapted to use in conventional equipment for immunoassays which are capable of detecting radioactivity, light adsorption in spectrophoto eters and light emission in fluoro eters 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.
- 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 the like.
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Abstract
On décrit de nouveaux procédés permettant d'analyser un analyte d'acide nucléique, qui consistent à utiliser des polynucléotidiques présentant des séquences oligonucléotidiques pratiquement homologues à la séquence à étudier dans l'analyte. La présence ou l'absence d'hybridation à une stringence prédéterminée permet de libérer un élément de marquage d'un support. En particulier différentes techniques sont utilisées pour fixer un élément de marquage à un support. Un clivage par un périodate d'une liaison entre l'élément de marquage et le support produit la libération de l'élément, lequel peut alors être détecté afin d'indiquer la présence d'une séquence oligonucléotidique particulière dans un échantillon. Le procédé peut être utilisé pour le diagnostic de maladies, la surveillance génétique et l'analyse de mélanges d'acides nucléiques.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US71667591A | 1991-06-17 | 1991-06-17 | |
US716675 | 1991-06-17 | ||
PCT/US1992/005099 WO1992022671A1 (fr) | 1991-06-17 | 1992-06-12 | Dosage de polynucleotides a l'aide de sites de clivage pouvant etre selectionnes |
Publications (1)
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EP0610215A1 true EP0610215A1 (fr) | 1994-08-17 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP92914302A Withdrawn EP0610215A1 (fr) | 1991-06-17 | 1992-06-12 | Dosage de polynucleotides a l'aide de sites de clivage pouvant etre selectionnes |
Country Status (6)
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EP (1) | EP0610215A1 (fr) |
JP (3) | JPH06509707A (fr) |
KR (1) | KR940701454A (fr) |
CA (1) | CA2110591A1 (fr) |
IE (1) | IE921940A1 (fr) |
WO (1) | WO1992022671A1 (fr) |
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DE19856796A1 (de) * | 1998-12-09 | 2000-06-15 | Biochip Technologies Gmbh | Spaltung von chemisch synthetisierten Oligo-/Polynucleotiden an einer vorbestimmten Stelle |
US6642000B1 (en) * | 1999-11-12 | 2003-11-04 | University Of Chicago | PCR amplification on microarrays of gel immobilized oligonucleotides |
WO2003064679A2 (fr) | 2002-01-30 | 2003-08-07 | Id Biomedical Corporation | Procedes pour detecter des microorganismes resistant a la vancomycine et composition pour la mise en oeuvre dudit procede |
CN103733047B (zh) | 2011-08-11 | 2016-03-30 | 奥林巴斯株式会社 | 目标粒子的检测方法 |
EP2752655A4 (fr) | 2011-08-30 | 2015-06-17 | Olympus Corp | Procédé de détection de particules cibles |
EP2818850B1 (fr) | 2012-02-22 | 2016-08-03 | Olympus Corporation | Procédé de détection d'une particule cible |
WO2013140890A1 (fr) | 2012-03-21 | 2013-09-26 | オリンパス株式会社 | Procédé de détection d'une molécule d'acide nucléique cible |
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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 (fr) * | 1987-10-15 | 1997-08-26 | Michael S. Urdea | Multimeres d'acide nucleique et essais d'hybridation amplifiee d'acide nucleique |
-
1992
- 1992-06-12 CA CA002110591A patent/CA2110591A1/fr not_active Abandoned
- 1992-06-12 WO PCT/US1992/005099 patent/WO1992022671A1/fr not_active Application Discontinuation
- 1992-06-12 KR KR1019930703922A patent/KR940701454A/ko not_active Application Discontinuation
- 1992-06-12 EP EP92914302A patent/EP0610215A1/fr not_active Withdrawn
- 1992-06-12 JP JP5501087A patent/JPH06509707A/ja active Pending
- 1992-07-01 IE IE194092A patent/IE921940A1/en not_active Application Discontinuation
-
1998
- 1998-11-30 JP JP10340645A patent/JPH11235198A/ja active Pending
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2002
- 2002-04-11 JP JP2002109760A patent/JP2002369699A/ja active Pending
Non-Patent Citations (1)
Title |
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See references of WO9222671A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1992022671A1 (fr) | 1992-12-23 |
IE921940A1 (en) | 1992-12-30 |
CA2110591A1 (fr) | 1992-12-23 |
JPH11235198A (ja) | 1999-08-31 |
KR940701454A (ko) | 1994-05-28 |
JP2002369699A (ja) | 2002-12-24 |
JPH06509707A (ja) | 1994-11-02 |
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