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WO1992000989A1 - Procede de marquage non-isotopique d'acide nucleique - Google Patents

Procede de marquage non-isotopique d'acide nucleique Download PDF

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Publication number
WO1992000989A1
WO1992000989A1 PCT/GB1991/001112 GB9101112W WO9200989A1 WO 1992000989 A1 WO1992000989 A1 WO 1992000989A1 GB 9101112 W GB9101112 W GB 9101112W WO 9200989 A1 WO9200989 A1 WO 9200989A1
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Prior art keywords
group
nucleic acid
protein
reactive
protein label
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PCT/GB1991/001112
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English (en)
Inventor
David Andrew Barstow
Andrew John Garman
John Rushington Parker
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Imperial Chemical Industries Plc
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Priority claimed from GB909015135A external-priority patent/GB9015135D0/en
Priority claimed from GB909018371A external-priority patent/GB9018371D0/en
Application filed by Imperial Chemical Industries Plc filed Critical Imperial Chemical Industries Plc
Publication of WO1992000989A1 publication Critical patent/WO1992000989A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention relates to a novel method for non-isotopic labelling of nucleic acids, in particular to the construction of DNA probes labelled with enzymes.
  • Current methods for the preparation of enzyme-labelled DNA probes fall into two types: those involving a covalent link between the enzyme and the probe, and those where the link is mediated via a specific binding pair such as avidin and biotin, or hapten and antibody.
  • This latter method is well suited to longer double stranded probes since it allows the introduction of the enzyme in the form of an enzyme binding partner conjugate, after the hybridisation of the probe to the conjugate has occurred. This means that there is no need to find denaturing conditions that keep the two strands of the probe apart and which do not interfere with enzyme activity.
  • DNA probes by reference to the DNA source (e.g. human genomic DNA) and the sequence of pairs of PCR primers, such that the scientist can readily perform the PCR reaction to obtain the desired probe.
  • DNA source e.g. human genomic DNA
  • sequence of pairs of PCR primers such that the scientist can readily perform the PCR reaction to obtain the desired probe.
  • probes would be labelled by use of radiolabelled nucleoside triphosphates.
  • biotin or hapten labelled nucleotides could be employed and the probe detected after hydridisation as described above.
  • An important aspect of the present invention is a method for the preparation of probes by an extension reaction, such as the PCR reaction, which are labelled with protein labels, for example, an enzyme, via a covalent linkage.
  • a preferred aspect of the invention therefore benefits from the advantages of the PCR method for defining and preparing DNA probes with the advantages of a covalent linkage between the label and the probe.
  • the method of the present invention is applicable to molecular biology research and to all areas of diagnostic medicine and other diagnostic sciences, for example forensic, agricultural, veterinary or food sciences where it is desirable to prepare probes for the detection or measurement of specific nucleic acid sequences.
  • it is applicable to the detection of infectious microorganisms and to the detection of point mutations, gene deletions and rearrangements which give rise to various inherited diseases and predispositions.
  • a method for the preparation of a protein labelled nucleic acid probe comprises performing an extension reaction on a nucleic acid template in the presence of one or more nucleoside triphosphates chemically modified for covalent linkage to a protein label, and covalently linking the resulting extended nucleic acid product with a reactive protein label.
  • nucleoside as used in this specification and in the claims includes deoxynucleosides.
  • extension on a nucleic acid template is meant any polymerase mediated extension of reaction whereby nucleotides can be incorporated into the growing nucleic acid strand. It includes known methods such as “primer extension” or “random hexamer labelling” (Feinberg A.P. and Vogelstein, Anal. Biochem 1983, 132,6 and Anal. Biochem. 1984, 137,266) and “nick translation” (Rigby P. .J. et al., J. Mol. Biol, 1977, 113,237) . It also includes RNA labelling systems such as those involving SP6 and T7 promoters (Melton D.A. et al., Nucl. Acid Res., 1984, ⁇ _ > 7035).
  • the extension reaction preferably includes terminally extending an oligonucleotide using one or more nucleoside triphosphates, which are chemically modified for covalent linkage to a protein label, in the presence of a suitable enzyme such as a terminal transferase.
  • a suitable enzyme such as a terminal transferase.
  • the template can be a single strand of nucleic acids which is itself extended.
  • the extension reaction is an amplification reaction ie. a reaction that can generate more than one copy of the sequence which is amplified.
  • the reactive protein label is preferably an enzyme, luminescent protein or a specific binding partner.
  • the protein is an enzyme.
  • Preferred enzymes are capable of generating a detectable signal, for example peroxidases such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, xanthine oxidase, and firefly or bacterial luciferase which have been chemically modified to make them reactive to a nucleophile such as an amino or thiol group.
  • peroxidases such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, xanthine oxidase, and firefly or bacterial luciferase which have been chemically modified to make them reactive to a nucleophile such as an amino or thiol group.
  • an enzyme that is stable to the commonly used methods for denaturing DNA for example boiling, are preferred.
  • protein labelled nucleic acid is intended to be hybridised with a target at high temperatures, e.g. 40-80°C
  • the enzyme is heat stable, for example stable in the range 40-80°C for a useful period of time, e.g. more than 30 minutes.
  • the desired stringency in hybridisations is achieved by denaturants such as formamide or urea.
  • Enzymes stable to such conditions in addition or instead of heat stability are likewise preferred.
  • Preferred methods of making reactive protein labels are described in our European Patent Application 431882.
  • a particularly convenient and preferred enzyme is alkaline phosphatase.
  • Luminescent proteins include the bioluminescent proteins such as obelin and aquorin, and the fluorescent proteins such as the phycobilliproteins.
  • Specific binding partners include avidin, streptavidin and specific monoclonal or polyclonal antibodies.
  • the amplification reaction is conveniently a PCR reaction as described above. Because of the need to denature double-stranded probes and because of the lability of most signal proteins to such denaturing conditions it is preferable that the PCR product be substantially single stranded, that is it contains sufficient single stranded material to give a probe of the required sensitivity without denaturation. Such substantially single stranded PCR products can be generated for example by performing a PCR reaction with one primer in excess over the other, as described for example by Gyllensten U. B. et al., P.N.A.S.,.1988, 5, 7652.
  • a standard 100 ul PCR reaction employing 100 pmole of a first primer and 2 pmole of a second primer in combination with a target nucleotide sequence will produce an excess of the strand generated by the first primer.
  • An alternative approach is to perform the PCR with one of the primers phosphorylated at the 5' end. After the PCR reaction the product is then treated with for example lambda exonuclease which digests the strand containing the 5' phosphate to give a single stranded product.
  • lambda exonuclease which digests the strand containing the 5' phosphate to give a single stranded product.
  • nucleic acid amplification methods based on polymerase-mediated extension reactions may be employed for example the transcription based system of Siska Diagnostics Inc., described in PCT patent application WO 88/10315. -
  • the amplification reaction employed in the method of the invention is the PCR reaction or one of its variants.
  • the amplification reaction may also be an enzyme mediated terminal extension reaction, such as any enzyme mediated extension reaction at one (or both) of the two termini of an oligonucleotide.
  • enzyme mediated terminal extension reaction such as any enzyme mediated extension reaction at one (or both) of the two termini of an oligonucleotide.
  • it includes forming a "tail" at the 3' end of the oligonucleotide in an extension reaction mediated by a terminal transferase enzyme.
  • this aspect of the invention specifically includes a method for the preparation of a protein labelled nucleic acid probe which method comprises performing an extension reaction mediated by a terminal transferase enzyme, preferably on the- 3' end of an oligonucleotide in the presence of one or more nucleoside triphosphates chemically modified for covalent linkage to a protein label, and covalently linking the resulting extended nucleic acid product with a reactive protein label.
  • the extension reaction preferably uses the 3' end of the previously formed oligonucleotide as an originating template and the terminal transferase catalyses terminal linking of any available nucleoside triphosphate.
  • the length and base composition cf the "tail” can be varied by appropriate adjustment of the rtaction conditions, in particular by varying the concentration of the reagents, especially terminal transferase and nucleoside triphosphate(s), and the reaction time.
  • the length of the "tail” can be assessed by, for example, gel eLectrophoresis.
  • the length of the tail will be long enough to ensure the incorporation of an adequate number of sites for covalent linking to a protein, but not so long that the "tail” interferes with hybridization of the probe section of the oligonucleotide.
  • the "tail” can be as short as one base but can be substantially longer.
  • the use of dideoxynucleoside triphosphate (chemically modified for covalent linking to a protein) as the nucleoside triphosphate(s) will prevent the extension reaction adding further bases (after the dideoxy base) because the dideoxy base residue lacks a 3'-hydroxyl group to act as the site for linking further bases to the nucleotide chain. This provides a convenient way of controlling the terminal transferase mediated extension reaction so as to extend the pre-formed oligonucleotide by a single base only.
  • This aspect of the invention is particularly valuable since it enables the preparation of oligonucleotide probes which are covalently linked or linkable to proteins which are, for example, an extension of an oligonucleotide which is known to be suitable as a probe for a particular application and in which the oligonucleotide probe in itself chemically unmodified as the modification is in the "tail".
  • the nucleoside triphosphate chemically modified for covalent linkage is preferably of the formula:
  • W is a nucleoside or dideoxynucleoside triphosphate
  • L is a linking group
  • X is an optionally protected chemically reactive group
  • nucleoside triphosphates there may be mentioned ATP, GTP, CTP, TTP and DTP, and analogues thereof. It will be understood that * where the extension reaction is a DNA extension reaction the group represented by W will be deoxynucleoside triphosphates.
  • the nature of the linker group L is designed to space the group X a sufficient distance from W such that (a) the compound of formula W - L - X is able to be recognised by a polymerase and incorporated in a growing nucleic acid chain during an ampli ication reaction, and (b) the compound of formula W - L - X, once incorporated in a nucleic acid chain, is preferably able to base pair to a complementary base, that is the hybridisation ability of the probe produced by the amplification reaction should preferably not be significantly affected.
  • the linker group L is preferably a divalent organic linker group of length 3 to 40 atoms, more preferably 6 to 30 atoms and especially from 10 to 25 atoms.
  • L is preferably an optionally substituted and optionally interrupted hydrocarbon chain.
  • Examples of preferred groups represented by L include: -(CH 2 ) 6 -,
  • the optional interruptions are incorporated in order to facilitate the synthesis of the chain and/or to provide more hydrophilic elements to maintain the linear structure of the chain and prevent it folding in on itself.
  • the site of attachment of the linker group L on the nucleoside triphosphate is most conveniently on the base moiety.
  • L should be joined to those atoms that are believed to be exposed in the major groove of the DNA double helix that is C5, C6 or the amino group on C4 of cytosine; C5, C6 or the oxygen attached to C4 of uridine; C5 methyl, C6 or the oxygen attached to C4 of thymine; N7, C8 or the amino group on C6 of adenine; N7, C8 or the oxygen attached to C6 of guanine.
  • X is a chemically reactive group which is not protected it is preferably a nucleophilic group or a group which is capable of being displaced by nucleophilic substitution to form a covalent bond between L and a nucleophile.
  • nucleophilic groups there may be mentioned groups of formula -NH , -SH, imidazolyl, -NRH (wherein R is optionally substituted alkyl, such as C -alkyl), -COOH, and anions of such groups.
  • X is a protected chemically reactive group it is preferably a protected nucleophilic group, especially a protected amino group or more preferably a protected thiol group; protecting groups for amino and thiol groups will be apparent to the organic chemist, and generally comprise a group which is stable under the normal aqueous conditions used for extension reaction, and yet can be removed chemically when desired.
  • Thiol and amino groups may be protected by any of the methods described in "Protective Groups in Organic Synthesis" by T.W.Green, Wiley Interscience. It is preferred that X is a protected thiol since we have found that there is a risk of unprotected thiols being oxidised by air or dimerising during the method of the invention. Thus modified nucleosides in which X is a protected thiol generally are more stable than the corresponding unprotected thiol, and this provides a particularly useful aspect of the invention. Examples of thiol protecting groups are given in Chapters 6 and 8 of T.W.Green's book, all of which are incorporated herein by reference thereto.
  • Preferred protected thiols represented by X are those which can be converted to a free thiol under conditions which do not degrade DNA or RNA, for example thio ethers, thio esters, disulphides and sulphenyl derivatives.
  • S-diphenylmethyl and S-triphenylmethyl thioethers such as S-diphenylmethyl, S-bis(4-methoxyphenyl)methyl, S-5-dibenzosuberyl, S-triphenylmethyl, S-diphenyl-4-pyridylmethyl, S-phenyl and S-2,4-dinitrophenyl methyl thioethers; S-t-butyl and S-1-adamantyl thio ethers; hemithio, dithio and aminothio acetals; substituted S-methyl groups such as S-methoxymethyl, S-isobutoxymethyl, S-2-tetrahydropyranyl, S-benzylthiomethyl, S-acetamidomethyl, S-benzamidomethyl, S-acetyl-, S-carboxy- and S-cyanomethyl thio ethers; substituted S-
  • Preferred thio esters represented by X include S-acetyl; S-benzoyl; S-thiobenzoyl; thiocarbonates such as S-2,2,2-trichloroethoxycarbonyl; S-t-butoxycarbonyl; S-benzyloxycarbonyl and S-p-methoxybenzyloxycarbonyl; and thiocarbamates such as S-(N-ethylcarbamate) and S-( - ⁇ tethoxybenzyloxycarbamate) .
  • Preferred disulphides represented by X include -S-S-ethyl, -S-S-t.butyl, and especially -S-S-aryl.
  • the -S-S-aryl groups, which form a further aspect of the invention, especially those which can act both as a protecting group and a reactive group are particularly preferred due to their stability during the extension reaction and their ability to react with nucleophilic groups in reactive protein labels to form a covalent bond with the protein.
  • Preferred -S-S-aryl groups are -S-S-(optionally substituted phenyl) groups such as -S-S-phenyl and -S-S-(3-carboxy-4-nitrophenyl); -S-S-pyridyl groups, especially -S-S-(2- or 4-pyridyl).
  • the -S-S-pyridyl and -S-S-(3-carboxy-4-nitro phenyl) are particularly preferred as cleavage of the -S-S-disulphide bridge produces a compound which is detectable by UV spectroscopy, thereby allowing successful cleavage to be determined.
  • Preferred sulphenyl derivatives represented by X include S-sulphonate and S-sulphenyl thio carbonate.
  • protected amino groups represented by X will be apparent to organic chemists, examples are fully described in Chapter 7 of the above mentioned book by T.W.Green and are incorporated herein by reference thereto.
  • Suitable protected amino groups include carbamates, amides, N-benzyl derivatives, imine derivatives, enamines and N-hetero 10
  • the resulting extended nucleic acid product may optionally be further modified, for example by addition of a thiol group (e.g. by reaction of an amino group with 2-iminothiolane) before covalently linking with the reactive protein label.
  • a thiol group e.g. by reaction of an amino group with 2-iminothiolane
  • X is an optionally protected sulphur group because it is found that thiols are more nucleophilic than amines and covalent linkage with reactive protein is thereby facilitated.
  • the protected thiol groups can be deprotected (i.e. converted to an -SH group) using appropriate deprotection conditions known to organic chemists such as those described in the above mentioned book by T.W.Green. For example, disulphides can be cleaved by mild aqueous reduction to give free thiols.
  • a particularly preferred modified nucleoside of the general formula W - L - X for use in the invention is shown in the free acid form by Formula (1) :
  • D is OH or preferably H
  • B is a divalent uracil, thymine, cytosine, adenine or guanine base
  • L is a linker group as hereinbefore described
  • X is -SH, -NHR or -S-P wherein R is H or C -alkyl, and P is a protecting group.
  • the reactive protein derivative is a protein label, as described above, which has in general been rendered reactive such that it is able to form a covalent bond with the group X on the amplification product.
  • Suitable pairs of reactive groups are known in the literature, for example in "Practice and theory of enzyme imraunoassays" by P.Tijssen, Elsevier, 1985 and in the Catalog of the Pierce Chemical Company.
  • the protein may be rendered reactive by derivatising its amino acid side chains, such as its amino groups, imidazole groups, guanidine groups, thiol groups (whether native or generated from disulphides by reductive cleavage) or carboxyl groups.
  • Glycoproteins may also be derivatised at the carbohydrate portion. The most convenient group to convert to a reactive group is the amino group.
  • the number of groups in the protein which are rendered reactive should preferably be kept low so as to minimise the chance of interfering with the protein's activity and to minimise the possibility of non-specific binding of the conjugate formed by the derivative. Therefore in general the number of reactive groups should be less than 5 moles per mole of protein, more preferably from 1 to 3 moles per mole of protein, most preferably 1 or 2 moles per mole of protein.
  • Maleimido is particularly useful.
  • Such groups may be attached to the protein by an optionally substituted saturated or unsaturated hydrocarbon chain containing for example up to 12, up to 10, up to 8, up to 6, up to 4 and conveniently up to 8 carbon atoms. Examples of optional substituents include hydroxyl groups.
  • the reactive protein derivative is in a stable, pre-activated form as described in our European Patent Application No.431882.
  • stable means that it can withstand commonly employed storage temperatures of for example -20°C, 4°C or ambient temperatures for useful periods of time, for example more than one month, preferably more than three months, most preferably more than six months, either in solution or in the lyophilised state. Such derivatives are usefully employed in a labelling kit using the method of the invention.
  • the conditions selected for covalently bonding the extended nucleic acid product with the reactive protein depend upon the nature of the extended nucleic acid product, e.g. the nucleophilicity of any groups represented by X, and the reactivity of the protein. In general the covalent binding will occur in an aqueous buffer of neutral or moderately alkaline pH at temperatures between 2 and 40°C. Optimum conditions for the covalent binding may be determined in individual cases by experiment. After the extended nucleic acid product and reactive protein have been covalently bonded there may be significant quantities of unreacted protein present together with other reactants or biproducts present. In order to reduce the non-specific binding properties of the probe mixture produced, it may in some circumstances be considered desirable J remove this unreacted protein and other impurities.
  • the method provides for an optional purification step after the conjugation step.
  • This may be achieved by any convenient means for example chromatography on a gel filtration, ion-exchange, hydrophobic, reverse phase or affinity column or ultrafiltration using a membrane of suitable pore size. It is preferred that any purification is brought about using a gel filtration column, conveniently a small disposable gel filtration column.
  • a particularly preferred group of the formula W - L - X is shown in its free acid form by Formula (2) wherein the encircled portion is a protecting group:
  • the compound of Formula (2) is hereinafter referred to as dUTP-21-SS-biptin.
  • the PCR reaction is preferably asymmetric, or alterna t ively the exonuclease version of the PCR as hereinbefore described.
  • Af tr - he PCR reaction is complete, and in the case of the exonuclease versiu.. of the PCR reaction, the exonuclease step has been performed, any protecting groups in the product may be removed.
  • W -L -X is of Formula (2)
  • the product is treated with a mild reducing agent, for example dithiothreitol, which cleaves the disulphide group thereby excising ' the encircled protecting group, and the mixture subjected to gel filtration, for example on Sephadex G25, which preferably gives a single stranded thiolated PCR product of the formula:
  • reaction of extended nucleic acid product and reactive protein label for example a protein label to.which maleimido groups have been attached, is allowed to proceed at approximately neutral or moderately alkaline pH for typically at least one hour after which the covalently bonded product may be purified by for example one of the methods described above.
  • E is a protein
  • L is a linker group
  • S is sulphur
  • K is, for example:
  • an assay method for detecting the presence, absence or amount of a target nucleic acid in a sample containing other nucleic acid sequences which are not sought to be detected comprises contacting the protein labelled nucleic acid of the present invention with the sample under hybridisation conditions, optionally removing unhybridised probe, and then detecting the presence or absence of hybridisation by means of the protein label.
  • the target nucleic acid may be from any source and is preferably of biological origin or an amplification product thereof. It is convenient to bind the target to a solid support such as a membrane, filter, bead or microtitre place well, either directly or via other binding moieties according to the nature of the assay.
  • a useful application is the detection of target nucleic acids bound to nylon or nitrocellulose filters arising from Southern blot, Northern blot, slot blot, dot blot, plaque lift or colony lift experiments.
  • a further use for the probe of the present invention is for the detection of nucleic acid sequences i ⁇ situ, i.e. in tissues.
  • hybridisation includes hybridisation between a polynucleotide probe and a desired target sequence but excludes hybridisation between a polynucleotide probe and non-desired nucleotide sequences. Suitable conditions for hybridisation are well known to the scientist of ordinary skill, see for example "Nucleic Acid Hybridisation", B. D. James and S. J.Higgins (Eds), IRL Press, Oxford, 1985.
  • kit of the invention comprises:
  • nucleoside triphosphate chemically modified for covalent linkage to a protein label
  • the preferred reactive protein label and chemically modified nucleoside triphosphate are as described above.
  • the kit preferably also contains one or more component selected from the following list: components for effecting the extension or amplification reaction, for example a polymerase or terminal transferase; a buffer for the amplification or extension reaction; the triphosphate of adenosine, guanosine, cytidine or thymidine; an exonuclease (for kits using the exonuclease method of generating single stranded PCR probes); components to effect the subsequent conjugation reaction, for example an agent for the deprotection of the chemically reactive group (where appropriate); a column for the removal of excess deprotection agent; reaction and column buffers; a column or other devices for the means of purification of the final conjugate; an agent for the further chemical modification on the resultant extended nucleic acid product e.g. 2-iminothiolane" and a protocol for carrying out the procedure according to the present invention.
  • components for effecting the extension or amplification reaction for example a polymerase or terminal transfera
  • Buffer A 0.1M triethanolamine-HCl pH 7.4, 1 mM MgCl ,
  • Buffer B 67 mM glycine - NaOH pH 9.4, 2.5 mM MgCl .
  • Buffer C 100 mM Tris-HCl pH 8.3, 500 mM KC1, 15 mM MgCl ,
  • Buffer H 0.1% BSA, 0.1% PVP, 0.1% Ficoll 400
  • Wash solution 2 0.25 x SSC, 0.5% SDS dUTP-21-SS-biotin; Purchased from Clontech, see Formula (2)
  • SSC 20x SSC is 3M NaCl, 0.3M trisodiur citrate
  • AP alkaline phosphatase
  • alkaline phosphatase (Boehringer, 10 mg/ml, 0.2 ml) was added 0.1M triethanolamine HC1, 1 mM MgCl , 1 mM ZnSO , pH 7.4 (0.6 ml), followed by 12 microlitres of a freshly prepared solution of SMCC (Pierce) in dry DMF (6.7 mg/ml) and the reaction mixture incubated at 25°C for 30 min.
  • the product was then purified by passage through a NAP 25 desalting column (Pharmacia), primed with BSA (Boehringer molecular biology grade) and equilibrated in PBS or, to prepare a stable lyophilised preparation, in 0.010 M sodium phosphate buffer pH 7.4 containing 10 g/1 D-lactose.
  • the product was collected in 1.6 ml and a portion taken for analysis.
  • Protein concentration was assessed by OD at 280 n (using an extinction coefficient of 0.89 for a 1 mg/ml solution) whilst the maleimido concentration was assessed as follows: 0.15 ml of sample was reacted with 10 microlitres of 1 mM mercaptoethanol for 30 min. at 37°C, alongside a control with 0.15 ml of buffer alone. The reactions were then diluted with 1.2 ml of PBS, zeroed at 412 nm in a spectrophotometer, and 25 microlitres of 1 mM 5,5', dithiobis(2-nitro-benzoic acid) added.
  • Remaining thiol concentrations were thereby measured using an extinction coefficient of 14150, the difference between sample and control enabling the maleimido concentration and hence the degree of substitution to be calculated. This value was found to be between 1.4 and 1.6 moles maleimido per mole of protein.
  • the derivatised enzyme was then aliquoted into 3 nmole portions and lyophilised. Preparation or oligonu ⁇ leotides
  • oligonucleotides were synthesised on an Applied Biosystems automated DNA synthesiser using protocols recommended by the manufacturer:
  • Primer 1 GGG CCT CAG TCC CAA CAT GGC TAA GAG GTG
  • Primer 2 CCC ACC TTC CCC TCT CTC CAG GCA AAT GGG
  • Primer 3 GAC TTC ACT TCT AAT GAT GAT TAT GGG
  • Primer 4 CTC TTC TAG TTG GCA TGC TTT GAT GAC GCT
  • Primer 2 was also synthesised with a 5' phosphate group prepared using the phosphoramidite "Phosphate ON” (Cruachem) using the manufacturer's protocol.
  • Human genomic DNA was amplified as follows: to an Eppindorf microfuge tube was added the dNTP mix (4 microlitres, 1 mM each), primers 1 and 2 (for amplification of alpha-1 anti-trypsin exon 3, 100 pmoles of each primer) or primers 3 and 4 (for amplification of cystic fibrosis gene exon 10, 100 pmoles of each primer), Buffer C (10 microlitres), human genomic DNA (30 ng in 6 microlitres, water to make up to 99 microlitres and 2.5 ⁇ of Taq polymerase (Perkin Elmer Cetus, 1 microlitre).
  • each tube was mixed, centrifuged and covered with light mineral oil (Sigma) and subjected to thermal cycling on a Perkin Elmer Cetus thermal cycler programmed as follows: 15 cycles of: 94°C, 2 minutes; 6 ⁇ "c, 2 minutes; 72°C,
  • Denaturation buffer was prepared as follows: to 1.48 ml of water was added 0.17 ml 1M Tris. HC1 pH 7.5, 0.30 ml 2M NaOH and 1.0 ml of 20x SSC. PCR product target (1 microlitre) was added to denaturation buffer (75 microlitres) and the samples incubated in a boiling water bath for 10 minutes, transferred to an ice bath and neutralised with 25 microlitres of 1M Tris. HC1.
  • a sample of human genomic DNA was amplified as described under "preparation of target” but with the following modifications: Primer 3 was used at 1 pmole, primer 4 at 100 pmole.
  • the concentration of dTTP was 20 micromolar.
  • the reaction included d ⁇ TP-21-SS-biotin at a concentration of 20 micromolar. 3 PCR reactions were performed and the products pooled.
  • dUTP- 1 -SS-biotin which is a nucleoside triphosphate chemically mo ⁇ * _ied for covalent linkage to a protein label
  • dUTP- 1 -SS-biotin which is a nucleoside triphosphate chemically mo ⁇ * _ied for covalent linkage to a protein label
  • the disulphide linkage was cleaved by incubating 25 microlitres of the PCR product for 10 minutes at room temperature with DTT to give a solution of -SH labelled DNA.
  • the solution was then passed through an NAP-25 desalting column to remove excess DTT, the -SH labelled DNA was eluted in 1.6 ml of Buffer A to give a DNA strand containing one or more units of Formula (3) and collected in a tube containing 3 nmoles of freeze-dried mal AP.
  • the -SH labelled DNA was reacted with mal AP by incubation therewith overnight at 4°C to form a covalent bond between AP and the sulphur atom of the -SH by displacement of maleic leaving groups present in mal AP.
  • the DNA labelled via the sulphur group to alkaline phosphatase is 'hereinafter referred to as the AP labelled CF probe.
  • the AP labelled CF probe was separated from free alkaline phosphatase and other impurities on a Sephacryl S-200 gel column (12 ml) equilibrated with PBS. Fractions were analysed by UV spectrometry and the first fractions, thereby shown to contain AP labelled CF probe, were pooled. The AP labelled CF probe was made up to a final volume of 2.2 ml including 0.1% BSA and 0.2% sodium azide.
  • CF slot blots prepared as described above under "Preparation of Slot Blots" were each probed with 0.5 ml of the above solution of AP labelled CF probe (1 nM) using the hydridisation procedure described above, and after the washes to remove unhybridised probe the membranes were treated with Lumi-Phos as described, and exposed to X-ray film for 5 minutes. A positive signal was observed with the 10 pg target CF DNA band. No signal was observed on a negative control (alpha 1 AT) slot which did not contain a CF DNA band.
  • Example 2
  • Primer 1 and 5' phosphoryl primer 2 were used (100 pmole each each) The concentration of dTTP was 20 micromolar. The reaction included dUTP-21-SS-biotin at a concentration of 20 micromolar. 2 PCR reactions were performed.
  • DNA from 170 microlitres PCR product was extracted once with phenol/chloroform (1:1, 170 microlitres), once with chloroform/isoamyl alcohol (24:1) and precipitated with ethanol (3 volumes).
  • the DNA was redissolved in 50 microlitres Buffer B and treated with 4 units of lambda exonuclease (obtained from BRL), for 15 minutes at 37°C to produce single stranded DNA in which at least one dUTP-21-SS-biotin unit had been incorporated.
  • DTT was added to a final concentration of 10 mM and the solution was incubated at room temperature for 10 minutes to cleave the disulphide bridge, thereby converting any dUTP-21-SS-biotin units in the DNA to a unit of Formula (2) having the free -SH group.
  • the resultant solution was passed through a NAP-5 desalting column (Pharmacia). The solution was eluted in 0.6 ml PBS and collected in a tube containing 125 microlitres of freshly prepared mal AP (approximately 1.8 nmols/ml).
  • the solution was mixed and incubated overnight at 4°C to a ⁇ low any -SH groups in the sample to form a covalent bond with alkaline phosphatase by nucleophilic displacement of maleic reactive groups.
  • the resultant DNA labelled via a sulphur group to alkaline phosphatase is hereinafter referred to as the AP labelled alpha 1 AT probe.
  • the AP labelled alpha 1 AT probe was separated from free alkaline phosphatase and other impurities on a Sephacryl S- 200 gel column eluted with PBS, eluting with-1.5 ml PBS, the volume was reduced to 1 ml on a speedy-vac concentrator and this was used to probe one alpha 1 AT slot blot, using the hybridisation procedure described above and a probe concentration of 1 nM.
  • step (iv) To a stirred solution of the product of step (iv) (0.45g, 0.92mmol) in dry pyridine (10ml) at 0 * C was added p-toluenesulphonyl chloride (0.19g, lmmol). Stirring was continued at 0 ⁇ C for 1 hour and the solution was allowed to warm to room temperature gradually. Stirring was maintained overnight, and then pyridine was removed by rotary evaporation. The residue was co-evaporated several times with toluene, redissolved in CHC1 /MeOH (9/1) and applied to a silica column. Elution with the same solvent gave the title compound as a white solid.
  • the mixture was diluted with water (5ml), applied to a column
  • oligomer of sequence 5'-GTGGATAGGGTGGATAGGGTGG (4nmoles) was incubated in a buffer containing 50mM KC1, lOmM Tris.HCl, 1.5mM MgCl , 0.01% gelatin, 0.1% Triton X-100, pH 9.0 in the presence of 20nmole of aminocaproyl dUTP and 75 units of terminal deoxynucleotidyl transferase in a total volume of 170-microlitres. The solution was incubated at 37°C for 2 hours. 18 microlitres of 3M sodium acetate and 470 microlitres of ethanol were added to the tube.
  • the tube was incubated at -70°C for 15 minutes and then spun in an Eppendorf microfuge for 15 minutes. The supernatant was discarded and the pellets were redissolved in 100 microlitres of 0.3M sodium acetate and 300 microlitres of ethanol was added to each. The tubes were again incubated at -70°C and spun in a microfuge. The resulting pellet was redissolved in water to give a final volume of 100 microlitres which was used directly in the next step.

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Abstract

Procédé de préparation d'une sonde d'acide nucléique marquée par une protéine, qui consiste à créer une réaction d'allongement sur une matrice d'acide nucléique en présence d'un triphosphate de nucléoside chimiquement modifié pour établir une liaison covalente, par exemple lorsqu'un bras de liaison d'une longueur de 3 à 40 atomes est terminé par un amino ou un thiol et rattaché de manière covalente à un marquage de protéine réactive. On décrit aussi des kits de diagnostic, un procédé d'analyse et des sondes d'acide nucléique. L'invention peut être utilisée en médecine, pour faire un diagnostic en médecine légale, et dans les sciences agricoles, vétérinaires et alimentaires.
PCT/GB1991/001112 1990-07-10 1991-07-08 Procede de marquage non-isotopique d'acide nucleique WO1992000989A1 (fr)

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GB9015135.8 1990-07-10
GB909015135A GB9015135D0 (en) 1990-07-10 1990-07-10 Novel nucleic acid labelling method
GB909018371A GB9018371D0 (en) 1990-08-21 1990-08-21 Novel nucleic acid labelling method
GB9018371.6 1990-08-21

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026932A1 (fr) * 1993-05-13 1994-11-24 United States Of America, As Represented By The Secretary, Department Of Health And Human Services Dosage immonologique marque a l'acide nucleique
WO1998005766A1 (fr) * 1996-08-02 1998-02-12 Bio Merieux Procede d'amplification d'une sequence d'un acide nucleique cible
WO1999041273A3 (fr) * 1998-02-11 1999-09-23 Perkin Elmer Corp Conjugues d'anp et d'adn et procedes de preparation desdits conjugues
WO1999063111A3 (fr) * 1998-06-01 2000-02-24 European Molecular Biology Lab Embl Methode de marquage d'acide nucleique
WO2000040590A3 (fr) * 1999-01-05 2000-11-02 Bio Merieux Compose fonctionnalise, polynucleotide eventuellement marque et procede de detection d'un acide nucleique cible
WO2000068422A3 (fr) * 1999-05-07 2002-04-04 Roche Diagnostics Gmbh Marquage haute densite d'adn par des nucleotides modifies ou portant un chromatophore et adn polymerases mises en application
EP0971039A3 (fr) * 1998-06-24 2004-01-28 Enzo Diagnostics, Inc. Méthode utiles pour l'amplification et pour le séquençage d'acides nucléiques, et la production d'acides nucléiques présentant une stabilité thermodynamique réduite
EP1466919A1 (fr) * 2003-04-05 2004-10-13 Roche Diagnostics GmbH Analogues nucleotidiques hexacycliques
US7276592B2 (en) 2003-04-05 2007-10-02 Roche Diagnostics Operations, Inc. Nucleotide analogs with six-membered rings
US8445664B2 (en) 1998-06-24 2013-05-21 Enzo Diagnostics, Inc. Kits for amplifying and detecting nucleic acid sequences
WO2017097973A1 (fr) * 2015-12-09 2017-06-15 Universitaet Konstanz Nucléosides modifiés

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EP0124221A1 (fr) * 1983-03-09 1984-11-07 Alan David Blair Malcolm Méthode de détermination d'une séquence de polynucléotide et polynucléotide marqué pour sa mise en oeuvre
US4587044A (en) * 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
WO1986002929A1 (fr) * 1984-11-08 1986-05-22 Life Technologies, Inc. Analogues de nucleotides pour le marquage et la detection de l'acide nucleique
US4772691A (en) * 1985-06-05 1988-09-20 The Medical College Of Wisconsin, Inc. Chemically cleavable nucleotides
WO1989002932A1 (fr) * 1987-10-02 1989-04-06 Cetus Corporation Conjugue covalent d'oligonucleotide-peroxidase de raifort

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EP0124221A1 (fr) * 1983-03-09 1984-11-07 Alan David Blair Malcolm Méthode de détermination d'une séquence de polynucléotide et polynucléotide marqué pour sa mise en oeuvre
US4587044A (en) * 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
WO1986002929A1 (fr) * 1984-11-08 1986-05-22 Life Technologies, Inc. Analogues de nucleotides pour le marquage et la detection de l'acide nucleique
US4772691A (en) * 1985-06-05 1988-09-20 The Medical College Of Wisconsin, Inc. Chemically cleavable nucleotides
WO1989002932A1 (fr) * 1987-10-02 1989-04-06 Cetus Corporation Conjugue covalent d'oligonucleotide-peroxidase de raifort

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Biochemistry, volume 28, 11 July 189, American Chemical Society, (Washington, D.C., US), M.M. Hanna et al.: "Synthesis and characterization of 5-Ä(4-azidophenacyl)thioÜuridine 5'-triphosphate, a cleavable photo-cross-linking nucleotide analogue", pages 5814-5820, see figures 1 and 2 *
Biotechniques, volume 8, no. 6, 1990, Eaton Publishing Natick, MA( US), J.J. Lanzillo: "Preparation of digoxigenin-labeled probes by the polymerase chain reaction", pages 620-622, see the whole article *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026932A1 (fr) * 1993-05-13 1994-11-24 United States Of America, As Represented By The Secretary, Department Of Health And Human Services Dosage immonologique marque a l'acide nucleique
US6537783B1 (en) 1996-08-02 2003-03-25 Bio Merieux Prefunctionalized nucleotide and process for amplifying a sequence using a prefunctionalized nucleotide
WO1998005766A1 (fr) * 1996-08-02 1998-02-12 Bio Merieux Procede d'amplification d'une sequence d'un acide nucleique cible
WO1999041273A3 (fr) * 1998-02-11 1999-09-23 Perkin Elmer Corp Conjugues d'anp et d'adn et procedes de preparation desdits conjugues
US6197513B1 (en) 1998-02-11 2001-03-06 Pe Corporation PNA and DNA conjugates and methods for preparation thereof
WO1999063111A3 (fr) * 1998-06-01 2000-02-24 European Molecular Biology Lab Embl Methode de marquage d'acide nucleique
US6764821B1 (en) 1998-06-24 2004-07-20 Enzo Life Sciences, Inc. Detecting the presence of specific target nucleic acid sequences through stem-loop formation
US8288092B2 (en) 1998-06-24 2012-10-16 Enzo Life Sciences, Inc. Modified nucleic acid polymers and methods for their production
EP0971039A3 (fr) * 1998-06-24 2004-01-28 Enzo Diagnostics, Inc. Méthode utiles pour l'amplification et pour le séquençage d'acides nucléiques, et la production d'acides nucléiques présentant une stabilité thermodynamique réduite
US6743605B1 (en) 1998-06-24 2004-06-01 Enzo Life Sciences, Inc. Linear amplification of specific nucleic acid sequences
US8486633B2 (en) 1998-06-24 2013-07-16 Enzo Diagnostics, Inc. Method for detecting nucleic acid sequences
US8445664B2 (en) 1998-06-24 2013-05-21 Enzo Diagnostics, Inc. Kits for amplifying and detecting nucleic acid sequences
US8133989B2 (en) 1998-06-24 2012-03-13 Enzo Diagnostics, Inc. Nucleic acid primer/construct compositions
US7264930B2 (en) 1998-06-24 2007-09-04 Enzo Biochem, Inc. Processes for non-linearly amplifying nucleic acids
US7713691B2 (en) 1998-06-24 2010-05-11 Enzo Life Sciences, Inc. Modified nucleic acid polymers and methods for their production
US7468245B2 (en) 1998-06-24 2008-12-23 Enzo Life Sciences, Inc. Methods for detecting nucleic acid sequences
US7485417B2 (en) 1998-06-24 2009-02-03 Enzo Life Sciences, Inc. Post-termination labeling of nucleic acid fragments and uses thereof
US6875858B1 (en) 1999-01-05 2005-04-05 Bio Merieux Functionalized polynucleotide compound, optionally marked and method for detecting a target nucleic acid
WO2000040590A3 (fr) * 1999-01-05 2000-11-02 Bio Merieux Compose fonctionnalise, polynucleotide eventuellement marque et procede de detection d'un acide nucleique cible
WO2000068422A3 (fr) * 1999-05-07 2002-04-04 Roche Diagnostics Gmbh Marquage haute densite d'adn par des nucleotides modifies ou portant un chromatophore et adn polymerases mises en application
US7276592B2 (en) 2003-04-05 2007-10-02 Roche Diagnostics Operations, Inc. Nucleotide analogs with six-membered rings
EP1466919A1 (fr) * 2003-04-05 2004-10-13 Roche Diagnostics GmbH Analogues nucleotidiques hexacycliques
WO2017097973A1 (fr) * 2015-12-09 2017-06-15 Universitaet Konstanz Nucléosides modifiés

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