EP0244471A1 - Mit lanthanidenchelat markierte nukleinsäureproben - Google Patents

Mit lanthanidenchelat markierte nukleinsäureproben

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Publication number
EP0244471A1
EP0244471A1 EP86907047A EP86907047A EP0244471A1 EP 0244471 A1 EP0244471 A1 EP 0244471A1 EP 86907047 A EP86907047 A EP 86907047A EP 86907047 A EP86907047 A EP 86907047A EP 0244471 A1 EP0244471 A1 EP 0244471A1
Authority
EP
European Patent Office
Prior art keywords
probe
nucleic acid
group
edta
formula
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Withdrawn
Application number
EP86907047A
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English (en)
French (fr)
Other versions
EP0244471A4 (de
Inventor
Gary Fred Musso
Soumitra Shankar Ghosh
Thomas Raymond Gingeras
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siska Diagnostics Inc
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Siska Diagnostics Inc
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Application filed by Siska Diagnostics Inc filed Critical Siska Diagnostics Inc
Publication of EP0244471A1 publication Critical patent/EP0244471A1/de
Publication of EP0244471A4 publication Critical patent/EP0244471A4/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis

Definitions

  • the present invention relates to nucleic acid hybridization probes. More particularly, it relates to probes chemically labeled with chelates of fluorescent lanthanide ions and to processes for making and using such probes.
  • a biological entity e.g., virus, microorganism, normal chromosome, mammalian chromosome bearing a defective gene
  • a biological entity e.g., virus, microorganism, normal chromosome, mammalian chromosome bearing a defective gene
  • the entity can be tested for using a nucleic acid probe.
  • a DNA or RNA associated with an entity to be tested for, and including a target sequence to which a nucleic acid probe hybridizes selectively in a hybridization assay, is called "target" DNA or RNA, respectively, of the probe.
  • a probe typically will have at least 8, and usually at least 12, ribonucleotides or
  • 2'-deoxyribonucleotides in the probing sequence that are complementary to a target sequence in target DNA or RNA.
  • the probe may have virtually any number and type of bases, as long as the sequences including these additional bases do not cause significant hybridization with nucleic acid other than target nucleic acid under hybridizaton assay conditions. That is, a probe will be specific for its target DNA or RNA in hybridization assays.
  • a polynucleotide probe To be useful in analyzing biological samples for the presence of target DNA or RNA, a polynucleotide probe must include a feature which will render detectable the duplex formed when the probe is hybridized to its complementary sequence in the target (single-stranded) DNA or RNA.
  • features in a probe include radioactive atoms, pyrimidine or purine bases chemically modified to include moieties (“tag moieties”) which can be detected by any of a number of techniques, or 5'-terminal phosphates similarly chemically modified.
  • a probe may be made with
  • 32 P-labeled nucleoside mono- or triphosphates can be detected by means of radiation from 32 P-decay.
  • Probes whose detectability is based on radioactive decay are unsuitable for many applications because of safety problems and licensing requirements associated with radioactive materials and because of degradation of the probes that occurs with radioactive decay during storage.
  • probes based on chemically modified nucleic acid there are numerous examples of probes based on chemically modified nucleic acid. Some of these chemically labeled probes are detected by means of fluorescent, luminescent, or other emissive or absorptive properties of the tag moieties themselves or chemical entities which occur observably (i.e., significantly above background) in a detection system only if tag moiety (and, consequently probe) is present. See e.g., Ward, et al., supra; Englehardt, et al., supra; Klausner and Wilson, supra; Heller, et al., European Patent Application Publication No. 0 070 687.
  • detection is, for example, by excitation of fluorescence from a fluorescent moiety, such as fluorescein, which is chemically linked directly to probe nucleic acid.
  • a fluorescent moiety such as fluorescein
  • a ligand such as biotinyl
  • detection is by fluorescence excitation of a fluorescent moiety, such as fluorescein, conjugated to a molecule, such as streptavidin or anti-biotin antibody in the case of biotinyl ligand, which binds tightly to the ligand when combined with probe in a hybridization assay.
  • probes detected by fluorescence employing techniques such as these, known heretofore, have a number of drawbacks.
  • sensitivity i.e., the minimum quantity of target nucleic acid that can be detected
  • this low sensitivity limits commercial applicability.
  • 100 to 1,000 times more target is required for detection with a probe detected by means of fluorescence than with a 32 P-labeled probe.
  • Probes dependent on enzymatic reactions to generate fluorescent compounds suffer from a need for long incubation periods for acceptable sensitivity in most applications.
  • Probes dependent on enzymes, antibodies or other complex biochemicals, such as streptavidin and biotin, for detectability suffer from the high cost of providing such materials with purity adequate for hybridization assays as well as the need for long incubation periods for detection.
  • the use of lanthanides as fluorescent tags in immunoassays has been reported. See Soini and Hemmila, U.S. Patent No. 4,374,120; Wieder and Wollenberg, U.S. Patent No. 4,352,751; Wieder (I), U.S. Patent No. 4,341,957; Wieder (II), U.S. Patent No.
  • nucleic acid hybridization probes can be labeled with tag moieties that chelate lanthanide ions, especially Eu(III), Tb(III), and Sm(III), and that thereby the fluorescent properties, as well as ease of use and low cost, of chelates of such ions can be exploited to overcome the various problems associated with other, particularly fluorescence-based, probe detection systems and provide probes of extraordinary sensitivity.
  • nucleic acid hybridization probes tagged with chelating agents of trivalent europium, terbium and samarium More specifically, we have discovered nucleic acid probes, DNA or RNA, labeled with polyaminocarboxylate derivatives that form chelates with high association constants with Eu(III), Tb(III), and Sm(III) in aqueous solution.
  • the probes of the invention are complexed with Eu +3 , Tb +3 or sm +3 and are detected by means of the intense fluorescence of these ions, particularly in chelates formed with aromatic trifluoromethyl ⁇ -diketones and synergistic Lewis bases that can readily be prepared in hybridization assay systems with probes of the invention.
  • Our invention also entails methods of making, and intermediates for use in making, probes of the invention and methods of using the probes in nucleic acid hybridization assays.
  • the probes of the invention are substantially improved over known probes, including in particular those detected by fluorescence. Detection of probes of the invention involves only inexpensive, stable, readily available chemicals and no enzymes, proteins or other complex and costly materials. Further, detection of probes of the invention is quite simple, involving no complex biochemical steps.
  • the probes of the invention involve no radioactive substances and none of the problems attendant with probes labeled or detected with such substances.
  • the sensitivity of probes of the invention is greater than that of known chemically tagged probes and is comparable to or greater than that of probes labeled radioactively to high specific activity.
  • nucleic acid probe DNA or RNA, which comprises a group of formula -F 1 L 1 F 2 R 1 , wherein -F 1 - and -F 2 - are functional groups at the termini of a linking moiety, -F 1 L 1 F 2 -, separated by a spacer group, -L 1 -, wherein -R 1 is a tag moiety that is a chelator of europium (III), terbium (III) or samarium (III), and wherein the group is bonded through -F 1 - to a nucleoside base of the probe, to a 5'-terminal nucleotide of the probe through the 5'-carbon of said 5'-terminal nucleotide, or to a 3'-terminal nucleotide of the probe through the 3'-carbon of said 3'-terminal nucleotide.
  • a tag moiety R 1 is linked to the 5'
  • the 5'-carbon of a 5'-terminal. nucleotide of a polynucleotide is referred to herein as the "5'-terminal carbon.”
  • the 3'-carbon of a 3'-terminal nucleotide of a polynucleotide is referred to herein as the "3'-terminal carbon.”
  • polynucleotide means any polymer of ribonucleotides or 2'-deoxyribonucleotides joined by 5'-3'- phosphodiester bonds and includes oligonucleotides as well as longer polymers. Usually all of the nucleotides of a polynucleotide will be either ribonucleotides or 2'-deoxyribonucleotides. However, in some cases, described below, a polynucleotide which otherwise consists of
  • 2'-deoxyribonucleotides might terminate with a ribonucleotide followed immediately, at the 3'-terminus, with a 2'-deoxyribonucleotide or a polynucleotide which otherwise consists of ribonucleotides might terminate with a 2'-deoxyribonucleotide.
  • group -F 1 L 1 F 2 R 1 is bonded to a 5'-terminal carbon of a probe of the invention
  • L 6 is alkyl of 3 to 20 carbon atoms
  • L 1 L 1 is typically (NH)L 2 or SCH 2 (CO)L 1 -,
  • the preferred linking moieties bonded to the 3'-terminal carbon are -OPO 2 NH(CH 2 ) n NH-, wherein n is 2 to 8.
  • the group (NH)- is represented herein as
  • CH 2 )- is represented herein as "OPO 2 SCH 2 " or -OPO 2 S(CH 2 )-”.
  • the 3'-terminal nucleotide of the probe will be a 2'-deoxyribonucleotide and the next nucleotide in the 5'-direction from said 3'-terminal nucleotide will be a ribonucleotide, regardless of whether the remainder of the probe is 2'-deoxyribonucleotides or ribonucleotides.
  • the group -F 1 L 1 F 2 R 1 When the group -F 1 L 1 F 2 R 1 is bonded to a nucleoside base of the probe, it will preferably be bonded to the 5-position of uracil moiety, although it can be bonded to other positions, including the 5-pos ⁇ tion or N 4 -nitrogen of a cytosme moiety and the
  • -CH CH(CO)(NH)L 1 -, - (CH 2 ) 2 (CO)(NH)L 1 -, and
  • -F 1 L 1 - is typically O, S or -NH-;
  • the tag moiety-chelating agent -R will preferably have a dissociation constant with Eu +3 , Tb +3 and Sm +3 in aqueous solution at 25°C between pH 5 and pH 9 that is less than 10 -17 M.
  • the preferred groups, R 1 for probes of the invention are EDTAyl, of formula: O
  • DTPAyl of formula: O
  • O p-EDTA-phenyl of formula: O
  • EDTA is an abbreviation for ethylenediaminetetraacetic acid.
  • DTPA is an abbreviation for diethylenetriaminepentaacetic acid.
  • probes of the invention include those wherein the tag moieties, R 1 , are complexed with
  • tag moiety R 1 is optionally complexed with Eu +3 , Tb +3 or Sm +3 .
  • a chelating group e.g., DTPAyl or EDTAyl or p-EDTA-phenyl
  • a compound of which the group is a part being “optionally complexed with Eu +3 , Tb +3 or Sm +3 " means that either the group chelates one of these lanthanide III ions or the group does not chelate any of the three lanthanide III ions.
  • the chelating group does not chelate Eu +3 , Tb +3 or Sm +3 , it might nonetheless, as the skilled will understand, be complexed with other metal ions, that might be present in solution with the chelating group, such as, for example, Na + or K + from buffers in the solution or magnesium, manganese, cobalt or other metal ions present in connection with enzymes.
  • the present invention includes a DNA or RNA probe which is made by a process which comprises reacting 1-(p-diazo-phenyl) EDTA, optionally (and preferably) complexed with Eu +3 , Tb +3 or Sm +3 , or a phenyl-azide-derivatized EDTA or
  • R263 is of formula
  • R 264 is H or n-alkyl of 1 to 3 carbon atoms, aa is 1 to 6, bb is 1 to 6 and cc is 0 or 1.
  • R 261 is optionally complexed with Eu +3 ,
  • R 261 , R 263 , R 264 , aa, bb and cc are novel and also an aspect of the present invention.
  • Reference herein to "phenyl azide-derivatized EDTA or DTPA" is, unless otherwise specifically qualified, to compounds of formula (R 263 )(NH)(CH 2 ) aa (NR 264 ) cc (CH 2 ) bb NH(R 261 ) as defined above in this paragraph.
  • the present invention entails also duplexes between probes of the invention and their respective target DNA's or RNA's. In another aspect, the present invention entails methods of making probes of the invention.
  • -CH CH(CO)(NH)- or a group terminated with a carbonyl group
  • -L 15 - is n-alkyl of 1 to 20 carbon atoms
  • -L 151 (NH)(CO)L 152 - or -L 151 (CO)(NH)L 152 - wherein -L 151 is bonded to F 15 and is n-alkyl of 1 to 17 carbon atoms.
  • In 52 is alk ⁇ l of 1 to 17 carbon atoms and L 151 and L 152 together have no more than 18 carbon atoms; and wherein, when -F 15 - is terminated with an amino group, -L 15 - is -CH 2 (CHOH)CH 2 O(CH 2 ) w OCH 2 (CHOH)CH 2 -, wherein w is 2 to 20, are known in the art. See, e.g., for enzymatic methods, Langer et al., supra; Ward et al., supra; Englehardt et al., supra; and Brakel et al., European Patent Application Publication No. 0 122 614.
  • the polynucleotide with free EDTAyl group (s) linked to pyrimidines is obtained by treating the polynucleotide (linked to EDTAyl-triester groups), after detachment from the solid phase, with glacial acetic acid and then isolating chromatographically and electrophoretically.
  • a probe of the invention with EDTAyl linked by the group of formula - (CH 2 ) 2 (CO)(NH)L 15 (NH)- to the 5'-carbon of pyrimidines and complexed with Eu +3 , Tb +3 or Sm +3 is obtained.
  • L 15 is preferably n-alkyl of 2 to 8 carbons and the EDTAyl is preferably linked to uracil moieties.
  • a polynucleotide (DNA or RNA) wherein one or more of the cytosines are modified to a moiety of formula
  • a nucleic acid with the sequence of the probe is reacted with hydrazine in the presence of bisulfite near neutral pH to convert a fraction of the amino groups bonded to carbon-4 of cytosines to hydrazine groups
  • F 17 is a suitably protected amino group
  • deprotection is carried out to yield an -NH 2 group from F 17 in groups bonded to the N -nitrogens
  • a polynucleotide which comprises a purine with a moiety of formula -F 18 L 18 NH 2 bonded to the carbon-8 position, wherein -F 18 - is O, S or NH and L 18 is n-alkyl of 1 to 20 carbon atoms, -L 181 (NH)(CO)L 182 - or -L 181 (CO)(NH)L 182 -, wherein -L 181 - is n-alkyl of 1 to 17 carbon atoms and is bonded to F 18 , -L 18 2 - is alkyl of 1 to 17 carbon atoms, and L 1 81 and L 182 together have no more than 18 carbon atoms, can be prepared by solid-phase, stepwise methods known in the art.
  • a polynucleotide which has the sequence of a probe and which comprises a pyrimidine moiety with a group of formula -F 15 L 15 NH 2 bonded to the carbon-5, a cytosine moiety with a group of formula -F 16 L 16 NH 2 bonded to the N 4 -nitrogen, or a purine moiety with a group of formula -F 18 L 18 NH 2 bonded to the carbon-8, wherein -F 15 , FX 16 , FX 18 ,
  • L 15 , L 16 and L 18 are as defined above, upon reaction with a suitable compound which includes tag moiety-chelator R 1 , and which is suitable for nucleophilic attack by the amino group at the terminus of the -F 15 L 15 NH 2 , -F 16 L 16 NH 2 or -F 18 L 18 NH 2 group will yield probe of the invention, wherein at least a fraction of the group or groups of formula -F 15 L 15 NH 2 , -F 16 L 16 NH 2 or -F 18 L 18 NH 2 on the polynucleotide are replaced with a group of formula -F 15 L 15 F 25 R 1 ,
  • DTPA anhydride Cho and Orgel, 1985, supra
  • PITCP-EDTA 1-(p-isothiocyanato-phenyl)EDTA
  • PICP-EDTA 1-(p-isocyanato-phenyl)EDTA, described below and hereinafter "PICP-EDTA”
  • EDTA and DTPA is also suitable for the reaction, provided that a water soluble carbodiimide coupling reagent, such as
  • reaction solution 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide, is present in the reaction solution.
  • Reaction with EDTA anhydride or DTPA anhydride is in aqueous buffer at a pH between 6 and 8 with the anhydride present at about 10 mg/ml and a 10-fold to 10,000-fold molar excess relative to polynucleotide.
  • Reaction with PITCP-EDTA or PICP-EDTA is in aqueous buffer at pH between 8 and 10 with the EDTA derivative in a 10-fold to 1,000-fold molar excess relative to nucleotide.
  • Reaction with EDTA or DTPA is in aqueous buffer at pH 6 to 7 with EDTA or DTPA at a 10-fold to 10,000-fold molar excess relative to nucleotide and carbodiimide at .01 M to .2 M and in large (10-1,000) molar excess relative to EDTA or DTPA.
  • the probe is isolated from the reaction mixture employing standard chromatographic procedures, particularly HPLC (high performance liquid chromatography) or gel permeation chromatography.
  • PITCP-EDTA, PICP-EDTA, or DTPA can be complexed with Eu +3 , Tb +3 or Sm +3 and used, in chelate form, in the nucleophilic reaction in essentially the same way as the unchelated form to make probe. Then, in the resulting probe, R, will be complexed with the lanthanide III ion.
  • probe that is complexed with the Eu +3 , Tb +3 or Sm +3 is prepared by the following procedure (referred to hereinafter as the "standard probe chelation process"): The lanthanide ion-free probe at between about 1 mg/ml and about 10 mg/ml in a volume of sodium citrate buffer, with citrate concentration between about
  • 0.05 M and about 0.5 M and pH of about 6.5 to about 7, is cooled on ice and is combined with an equal volume of a solution, in HCl at about 0.1 M to about 1 M (about twice the concentration of citrate in the probe solution), of a salt of the lanthanide ion, with a concentration of said salt between about 0.1 times equimolar and between about 25 times equimolar, preferably about 1 time to 2 times equimolar, with respect to the concentration of chelator tag moieties R 1 linked to probe in the solution.
  • the pH of the resulting solution is adjusted if necessary to about 3 to about 3.5 by addition of NaOH or HCl and incubated on ice for about 10 to about 20 minutes.
  • the pH of the solution is increased to neutral (i.e., 6 to 8) by additon of 1 M of NaOH and the solution is briefly
  • the labeled probe, complexed with the Eu +3 , Tb +3 or Sm +3 is isolated from the solution by a standard procedure, e.g., by gel filtration using Sephadex G-50 with 0.1 M to 0.5 M sodium citrate (pH 6.5 to 7).
  • Preferred salts for this purpose are EuCl 3 ,
  • a DNA can be prepared by employing E.
  • dUTP and dCTP are known compounds or are readily prepared by the skilled employing known techniques. See, e.g., Ward et al., supra.
  • UTP and CTP analogs like their dUTP and dCTP counterparts, are known compounds or are readily prepared by the skilled.
  • TdT deoxynucleotidyl transferase
  • the group R 26 on the modified dUTP is optionally, and preferably, complexed with Eu +3 , Tb +3 or Sm +3 ; the preferred groups bonded to carbon-5 of the modified dUTP or dCTP employed in the extension reaction are
  • the preferred TdT is from calf thymus.
  • metal ions such as Mg +2 , Mn +2 or Co +2 must be present for enzymatic activity, as known in the art.
  • Mg +2 , Mn +2 , Co +2 must be present or the TdT will not catalyze extension of said strand.
  • These metal ions e.g., Mg +2 , Mn +2 , Co +2 , are chelated by tag moiety-chelators of formula -R 261 or R 262 .
  • the group R 26 linked to the modified UTP, CTP, dUTP or dCTP employed in the enzymatic reaction is not complexed with metal ion, it will chelate metal ion that must be present in the enzyme reaction mixture for enzymatic activity.
  • probe to be made by one of the above-described enzymatic reactions is intended to have tag moiety not complexed with metal ion
  • the probe isolated from the reaction mixture must be treated to separate metal ion from the tag moieties. This can be accomplished, for example, by dialyzing solution with the probe against metal-free buffer using standard procedures known in the art.
  • UTP, CTP, dUTP or dCTP used in the enzyme reaction was complexed with Eu +3 , Tb +3 or Sm +3 , will be treated by the standard probe chelation process described above.
  • R 26 is R 261 (i.e., EDTAyl or DTPAyl)
  • R 26 is R 261
  • EDTA or DTPA can be reacted directly with the
  • -CH CH(CH 2 ) v NH 2 -derivatized ribonucleotide or 2'-deoxyribonucleotide in the presence of a water-soluble carbodiimide coupling reagent, such as 1-ethy1-3-(3-dimethylaminopropyl) carbodiimide, at about pH 6 to 7, with the carbodiimide at about 0.01 M to 0.2 M and large molar excess relative to both nucleotide and EDTA or DTPA.
  • a water-soluble carbodiimide coupling reagent such as 1-ethy1-3-(3-dimethylaminopropyl) carbodiimide
  • -R 26 is p-EDTA-phenyl
  • the PICP-EDTA is prepared following the procedure of Hemmila et al. (1984), supra, for preparation of PITCP-EDTA by condensing PDP-EDTA in a water-chloroform mixture with phosgene, removing the aqueous layer, and isolating the PICP-EDTA from the aqueous layer by drying.
  • EDTA and DTPA chelates of Eu +3 , Tb +3 and Sm +3 are known.
  • PITCP-EDTA complexed with Eu +3 is known (see Hemmila et al. (1984), supra). This compound complexed with Tb +3 or Sm +3 is made in the same way as the Eu +3 complex except that TbCl 3 or SmCl 3 is employed in place of EuCl 3 .
  • the lanthanide ion complexes of PICP-EDTA are prepared in the same way as the lanthanide ion complexes of PITCP-EDTA.
  • any of the methods described below for preparing a double-stranded DNA which comprises a DNA with sequence of a probe can be applied to provide a double-stranded DNA template for use in the above-described methods for preparing, by DNA polymerase-, RNA polymerase- or TdT-catalyzed nucleic acid synthesis, a probe of the invention comprising a modified uracil or cytosine moiety.
  • the methods described below for preparing a single-stranded DNA with sequence of a probe can be used to supply a single-stranded DNA substrate for preparation with TdT of a probe of the invention comprising a modified uracil or cytosine moiety.
  • one method of the invention for making a probe of the invention comprises providing a precursor polynucleotide, which is a polynucleotide which has the sequence of the probe and which comprises a nucleoside base bonded to a linker moiety of formula -F 1 L 1 NH 2 and (i) reacting said polynucleotide with a compound selected from EDTA anhydride, DTPA anhydride, PITCP-EDTA or PICP-EDTA, wherein the PITCP-EDTA or PICP-EDTA is optionally complexed with Eu +3 , Tb +3 or Sm +3 or (ii) in aqueous solution buffered to a pH of 6 to 7, reacting said polynucleotide with EDTA or DTPA, wherein the EDTA or DTPA is optionally complexed with Eu +3 , Tb +3 or Sm +3 , with a water soluble carbodiimide coupling agent.
  • any water soluble carbodiimide coupling agent known in the art can be employed, such as, for example,
  • 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide Numerous methods of providing the polynucleotide are available, as described above. If the probe obtained by one of the above reactions is not complexed with Eu +3 , Tb +3 or Sm +3 , a probe that is so complexed is obtained, usually after purification by HPLC or gel permeation chromatography, by carrying out the above-described standard probe chelation process with the uncomplexed probe. If DTPA, EDTA, PICP-EDTA or PITCP-EDTA complexed with lanthanide ion is employed in.
  • the eluant employed in chromatographic isolation of the resulting probe will include preferably sodium citrate at about 0.1 M-0.5 M and pH 6.5 to 7 or, alternatively, DTPA (or EDTA) at about 10 um-100 uM with CaCl 2 at about twice the DTPA or EDTA concentration, in order to remove from purified probe any lanthanide ion freed during the reaction and not complexed with EDTA or DTPA tag moiety on the probe.
  • the group -F 1 L 1 NH 2 is preferably bonded to the 8-position of a purine moiety, wherein it is preferably of formula -NH(CH 2 ) t NH 2 wherein t is 2 to 8, or to the
  • R 26 is optionally complexed with Eu +3 , Tb +3 or
  • a DNA segment extended in the reaction preferably comprises, prior to the reaction, a probing sequence suitable for the target DNA or RNA of the probe, although such a probing sequence can be made in the extension reaction.
  • a probing sequence suitable for the target DNA or RNA of the probe, although such a probing sequence can be made in the extension reaction.
  • Preferably only modified dUTP or dCTP (or both) will be employed as substrate in the extension reaction, and the reaction will be carried out so that, on the average, 1 to 5 modified nucleotides are added to the 3'-terminus of each extended polynucleotide.
  • the method can be employed advantageously with single-stranded DNA, from an automated synthesizer, that is about 12 to about 100 nucleotides long.
  • nucleic acid probe according to the invention wherein the DNA or RNA is non-specifically labeled with p-EDTA-phenyl, complexed with Eu +3 , Tb +3 or Sm +3 if the PDP-EDTA was, as preferred, so complexed, as a result of the nucleophilic displacement by nucleophiles on the polynucleotide of N 2 from the diazo phenyl of the PDP-EDTA under neutral to alkaline conditions.
  • EDTAyl or DTPAyl is optionally (and preferably) complexed with Eu +3 , Tb +3 or Sm +3 , R 263 is R 264 is hydrogen or n-alkyl of 1 to 2
  • aa is 1 to 6
  • bb is 1 to 6
  • cc is 0 or 1 is reacted under photoactivating conditions with a polynucleotide with a sequence of a probe.
  • This process yields a nucleic acid probe according to the invention wherein the DNA or RNA is non-specifically labeled as a result of reaction with the nitrene which results from photolysis of the azide. If the phenyl azide derivative employed in the reaction was complexed with Eu +3 , Tb +3 or Sm +3 , the probe resulting from the reaction will be so complexed as well.
  • Photoactivating conditions simply require that the solution of polynucleotide with sequence of the probe and of phenyl-azide-derivatized EDTA or DTPA (optionally complexed with Eu +3 , Tb +3 or Sm +3 ) be illuminated with light of wavelength low enough to photolyze the phenyl azide to a phenyl nitrene and preferably high enough to avoid damage to the polynucleotide ultraviolet light. Wavelengths between about 340 nm and 380 nm are suitable.
  • Example XI EDTA's and DTPA's of the invention is illustrated in Example XI with the compound wherein R 261 is DTPAyl,
  • R 264 is _ CH 3 , aa is 3, bb is 3, and cc is 1.
  • the phenyl azide-derivatized DTPA or EDTA can be complexed with Eu +3 , Tb +3 or Sm +3 by the same method as PDP-EDTA, but carried out in the dark.
  • single-stranded polynucleotide is preferably employed.
  • the process is illustrated in Example V for PDP-EDTA and Example XII for phenyl azide-derivatized EDTA or DTPA.
  • the process is carried out with an initial molar concentration of PDP-EDTA, or phenyl-azide- derivatized EDTA or DTPA, of between about 0.1 X and 2 X the molar concentration of deoxyribonucleotides or ribonucleotides in the polynucleotide with sequence of probe that is to be labeled in the reaction.
  • any of the processes described below for providing a polynucleotide with sequence of probe can be employed to provide polynucleotide to be labeled by the process of reacting with PDP-EDTA, optionally and preferably complexed with Eu +3 , Tb +3 or Sm +3 , or with phenyl-azide- derivatized EDTA or DTPA of formula (R 263 )NH(CH 2 ) aa (NR 264 ) cc (CH 2 ) bb NH(R 261 ), wherein R 261 ,
  • R 263 , R 264 , aa, bb and cc are as defined above and the compound is optionally and preferably complexed with Eu +3 , Tb +3 or Sm +3 .
  • the reaction is carried out by combining an aqueous solution of polynucleotide, preferably single-stranded, at between about 0.001 mg/ml and 3 mg/ml concentration, with an aqueous solution of the PDP-EDTA or phenyl-azide-derivatized EDTA or DTPA, at between about 0.3 uM and 2 mM (about 0.1 X to 2 X the molar concentration of nucleotides) and allowing the reaction to proceed at 0°C to 10°C for between about 1 hour and 8 hours at a pH between about 7.5 and 8.5 (with PDP-EDTA) or about 6 and 8 (with the phenyl azide-derivatized EDA or DTPA).
  • the reaction with phenyl-azide-derivatized EDTA or DTPA occurs under photoactivating conditions.
  • the probe if the reaction was run with PDP-EDTA or phenyl azide-derivatized EDTA or DTPA, not complexed with Eu +3 , Tb +3 or Sm +3 , is purified from the reaction mixture (a) chromatographically, preferably by gel permeation chromatography on, for example. Sephadex G-50, using a buffer such as 0.01 M Tris-HCl at a pH between about 7 and about 8 as eluant or (b) by precipitation, as with ethanol.
  • the chromatographic purification of probe will be by gel permeation chromatography employing, for example, Sephadex G-50 and 0.1 M to 0.5 M sodium citrate, pH 6.5 to 7, as eluant.
  • the citrate eluant serves to complex any dissociated lanthanide ion and separate it from probe being purified.
  • An alternative, but less preferred, eluant to accomplish this purpose of separating dissociated lanthanide ion from probe is about 10 uM to about
  • the probe obtained from the reaction between PDP-EDTA, or phenyl-azide-derivatized EDTA or DTPA, and polynucleotide is, after purification by chromatography or precipitation as described above, subjected to the standard probe chelation process with a salt of Eu +3 , Tb +3 or Sm +3 .
  • the reaction between PDP-EDTA, or phenyl azide-derivatized EDTA or DTPA, and polynucleotide is carried out so that between about 1 in 12 and about 1 in 1,000, most preferably about 1 in 100, nucleotides in the probe is labeled.
  • the extent of labeling under given reaction conditions can be determined by spectroscopic and other analytical techniques well known in the art and reaction conditions can be adjusted appropriately to achieve a desired extent of labeling.
  • the extent of labeling can be determined by forming a lanthanide III ion (e.g., Eu +3 ) complex with the non-specifically labeled polynucleotide and then measuring the amount of chelated lanthanide III ion by extracting, from a known quantity of the labeled polynucleotide, the ion employing a fluorescence enhancement solution, described below, and comparing the fluorescence intensity from the resulting solution with that from comparable standard solutions which have known concentrations of the lanthanide ion.
  • a lanthanide III ion e.g., Eu +3
  • phenyl azide-derivatized compounds between about 1% and 3% of the phenyl azide derivative in solution reacts with polynucleotide. See, e.g., Staros, Trends in Biochemical Sciences 5, 320-322 (1980); and
  • the methods of the invention for preparing probe by non-specific reaction with PDP-EDTA (optionally complexed with Eu +3 , Tb +3 or Sm +3 ), or phenyl azide-derivatized EDTA or DTPA (also optionally complexed with Eu +3 , Tb +3 or Sm +3 ), is preferably carried out with probes between about 100 and 10,000 nucleotides in length.
  • nucleic acid probe of the invention uses as starting material a nucleic acid (DNA or RNA) with sequence of probe which has: (i) a 5'-terminal carbon bonded to a group of formula
  • L 5 is alkyl of 2 to 20 carbon atoms
  • L 6 is alkyl of 3 to 20 carbon atoms
  • nucleic acids with modified terminal nucleotides are preferably employed to make probes, between about 10 and about 100 nucleotides in length, which are based on nucleic acids that can be synthesized advantageously by automated, stepwise solid phase methodology.
  • the more preferred of the methods employ nucleic acids with modified 5'-terminal nucleotides.
  • a nucleic acid with a 5'-terminal nucleotide modified to have a group of formula -OPO 2 (NH)L 5 NH 2 bonded to the 5'-carbon can be prepared by the methods of Chu et al., Nucleic Acids Research 11, 6513-6529 (1983); see also Chu and Orgel, Proc. Natl. Acad. Sci. 82, 963-967 (1985). The methods of Chu et al. (1983), supra, and Chu and Orgel (1985), supra, can also be employed to prepare a nucleic acid with a group of formula -OPO 2 (NH)L 5 NH 2 bonded to the 3'-carbon of the 3'-terminal nucleotide.
  • the single-stranded nucleic acid with the desired sequence and with a phosphate group bonded to the 3'-terminal carbon or the 5 '-terminal carbon is provided.
  • This nucleic acid is then reacted for 2-4 hours at room temperature in the presence of approximately 0.1 M imidazole-HCl buffer (about pH 6) and approximately 0.1 M of a water soluble carbodiimide coupling agent, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, to form the phosphoroimidazolide derivative.
  • a water soluble carbodiimide coupling agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • the phosphoroimidazolide derivative is isolated by HPLC and is then reacted for 2-4 hours at 50°C and at a pH between about 7 and about 8 with a diamine of formula H 2 NL g NH 2 , at a concentration of between about
  • the nucleic acid with the 3'-terminal carbon or 5'-terminal carbon bonded to a phosphate group is combined with 0.05 M to 0.5 M diamine of formula H 2 NL 5 NH 2 , approximately 0.1 M methylimidazole.HCl buffer (about pH 6) and approximate 0.1 M of a water soluble carbodiimide coupling agent, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, the mixture is incubated for 12-20 hours at room temperature, and the desired polynucleotide, derivatized at the carbon with a group of formula -OPO 2 (NH)L 5 NH 2 , is purified by HPLC.
  • L 5 is preferably n-alkyl of 2 to 8
  • the methods could be applied to a mixture of polynucleotides, some with 5'-terminal-5'-phosphates, some with 3'-terminal-3'-phosphates, and some with both 5'-terminal-5'-phosphates and 3'-terminal-3'-phosphates, resulting from. random cleavage of polynucleotide, as by sonication.
  • the preferred phosphate-terminated polynucleotides for use in the invention are those with phosphate bonded to the 5'-terminal carbon.
  • polynucleotide with the desired sequence of probe by an automated, stepwise, solid phase synthesis procedure and then 5'-phosphorylating the polynucleotide using standard procedures with T4 polynucleotide kinase.
  • Polynucleotides phosphorylated with T4 polynucleotide kinase will have 3'-terminal nucleotides with hydroxylated 3'-carbons and thus can be employed to make probe with TdT, with T4 RNA ligase, or with TdT followed by T4 RNA ligase as described elsewhere herein.
  • a nucleic acid with a group of formula -OPO 2 SCH 2 (CO)L 5 NH 2 bonded to the 5'-terminal carbon is prepared in two steps.
  • T4 polynucleotide kinase-catalyzed reaction Conditions for this T4 polynucleotide kinase-catalyzed reaction are the same as the known conditions that would be employed if ATP were the substrate.
  • nucleic acid so modified is reacted with an alpha-haloketone derivative of formula H 2 NL 5 (CO)CH 2 X 5 , wherein X 5 is chloro or bromo, under conditions known to, or readily ascertained by, the skilled to be suitable for nucleophilic displacement of the halogen by the sulfur of the thiophosphate.
  • the compounds of formula H 2 NL 5 (CO)CH 2 X 5 are known or readily synthesized by the skilled using known methods.
  • -OPO 2 SCH 2 (CO)L 5 NH 2 bonded to the 3'-terminal carbon is prepared in either of two ways, based on modifications of the teaching of Cosstick et al., Nucl. Acids Rsch. 12, 1791-1800 (1984). Both of the methods employ the known enzyme T4 RNA ligase and, as nucleic acid substrate, a polynucleotide with a ribonucleotide at its 3'-terminus, said ribonucleoside having a hydroxyl group bonded to its 3'-carbon.
  • a polynucleotide can be either RNA or DNA with such a ribonucleoside at its 3'-terminus.
  • a DNA with a hydroxyl bonded to its 3'-terminal carbon can be ligated, through said hydroxyl, to a ribonucleoside-5'-phosphate in a reaction catalyzed by TdT.
  • TdT a reaction catalyzed by TdT
  • Cosstick et al. is ligated to the 3'-terminus of the polynucleotide with the 3'-terminal ribonucleoside in a reaction catalyzed by T4 RNA ligase. Then, the resulting polynucleotide, with the group of formula O 2 H bonded to the 3'-carbon of the 3'-terminal
  • the 2'-deoxyribonucleoside-5'-phosphate-3'- thiophosphate is dissolved to give a 1 uM to 10 uM solution in .05 M aqueous HEPES, pH 7.
  • To 1 ml of the solution is added with stirring 10-20 ul of an acetonitrile solution that is 1 mM in compound of formula H 2 NL 5 (CO)CH 2 X 5 . Stirring is continued at room temperature for 1 hour.
  • the solution is then diluted to 4 ml with water and the desired product isolated chromatographically.
  • EDTA anhydride, DTPA anhydride, or EDTA or DTPA not complexed with Eu +3 , Tb +3 or Sm +3 is subjected to the standard probe chelation process; or the product from reaction with EDTA or DTPA complexed with Eu +3 , Tb +3 or Sm +3 , is purified using 0.1-0.5 M sodium citrate, pH 6.5-7, as eluant in the chromatography.
  • PITCP-EDTA or PICP-EDTA is not so complexed and the product is isolated chromatographically.
  • the product of the reaction is subjected to the standard probe chelation process.
  • the chromatographic purification of product employs 0.1 M-0.5 M sodium citrate, pH 6.5-7 as eluant.
  • novel 3'-thiophosphate adducts of the 5'-phosphate -2'-deoxyribonucleoside, wherein the group of formula -OPO 2 SCH 2 (CO) L 5 F 28 R 28 is bonded to the 3'-carbon is another aspect of our present invention, as are the various salts (e.g., with alkali metal ions or Mg +2 ) , acid and base forms, and hydrates of the novel compounds, all of which can be prepared easily by the skilled.
  • the adducts are substrates for the T4 RNA ligase.
  • L 5 be n-alkyl of 2 to 20 carbon atoms, and most preferred that L 5 be n-alkyl of 4 to 6 carbon atoms.
  • -OPO 2 SCH 2 (CO)L 51 F 28 R 28 can also be bonded to the 3'-terminal carbon of the polynucleotide, wherein L 51 is the same as or different from L 5 and is alkyl of 2 to 20 carbons and wherein the group of formula -OPO 2 NH- or -OPO 2 S-, bonded directly to the
  • 5'-terminal carbon need not be the same as the group, of formula -OPO 2 NH- or -OPO 2 S-, bonded directly to the 3'-terminal carbon.
  • a nucleic acid with a desired sequence and with an amino group (-NH 2 ) bonded to the 5'-terminal carbon is prepared by the method of Smith et al., Nucl. Acids Research 13, 2399-2412 (1985). The method is preferably carried out on an automated synthesizer, such as the Model 380A of Applied Biosystems, Inc. (Foster City, California, U.S.A.). The method of Smith et al. (1985), supra, entails application of the phosphoramidite chemistry of Matteucci and Caruthers, J. Am. Chem. Soc. 103, 3185 (1981), and Beaucage and Caruthers, Tetrahedron Lett.
  • the polynucleotide with the 5'-amino-group on the 5'-terminal nucleotide is obtained.
  • the -OPO 3 L 6 SH- derivatized polynucleotide is reacted with a mixed disulfide of formula R 5 -S-S-L 5 -NH 2 , wherein R 5 is 2-pyridyl or 4-pyridyl, to yield the polynucleotide with a group of formula -OPO 3 L 6 SSL 5 NH 2 bonded to the 5'-terminal carbon.
  • This polynucleotide is then purified by known chromatographic procedures (e.g., HPLC).
  • S-trityl phosphite derivatives of mercaptoethanols are known compounds, as taught by Connolly and Rider, supra.
  • the result is a resin-bound polynucleotide with a group of formula -L 6 -S-C(C 6 H 5 ) 3 bonded to the 5'-terminal carbon.
  • the polynucleotide is treated with thiophenolate to remove phosphate protecting groups and then ammonia to remove base protecting groups and cleave polynucleotide from the solid support.
  • the polynucleotide, with the S-trityl bond intact, is isolated by HPLC.
  • the polynucleotide is treated with a 5-fold molar excess (relative to polynucleotide) of silver nitrate followed, after 30 minutes, with a 7-fold molar excess of dithiothreitol.
  • the treatment with silver ion cleaves the S-trityl bond.
  • the treatment with dithiothreitol is to remove silver ion. After 30 minutes, the precipitated silver salt of dithiothreitol is removed by centrifugation.
  • the desired, derivatized oligonucleotide remains in the supernatant and is isolated and purified from the supernatant by HPLC, and is then reacted with R 5 -S-S-L 6 -NH 2 in a mixture of acetonitrile/water for 16 hours at 23°C, as described aoove, to finally obtain the desired polynucleotide, derivatized with -OPO 3 L 6 SSL 5 NH 2 , which is isolated by chromatography over Sephadex G-50.
  • the probe is purified from the reaction mixture by gel permeation chromatography, as, for example, on Sephadex G-50, using a buffer such as 0.01 M Tris-HCl at a pH between about 7 and about 8, as eluant; the standard probe chelation process is then used to complex Eu +3 , Tb +3 or Sm +3 to the probe when desired.
  • the reaction is optionally, and preferably, carried out with the PITCP-EDTA or PICP-EDTA complexed with Eu +3 , Tb +3 or Sm +3 ; if the reaction is so carried out, the eluant in the gel permeation chromatography purification will preferably contain about 0.1 M to 0.5 M sodium citrate and be at pH 6.5 to 7.
  • the probe of the invention resulting from reaction with PITCP-EDTA will have a group of formula
  • the probe resulting from reaction of said reagent with a nucleic acid with the sequence of probe and with the 5'-terminal carbon bonded to an amino group or a group of formula -OPO 2 (NH)L 5 NH 2 will have, linked to said 5'-carbon as indicated above, a p-EDTA-phenyl group that is complexed with said Eu +3 , Tb +3 or Sm +3
  • EDTAyl is bonded to the 5'-terminal carbon by reaction of nucleic acid, with a group of formula
  • DTPAyl or EDTAyl-derivatized nucleic acid combine it with a solution of Fe +2 , and thereby convert the
  • DTPAyl or EDTAyl groups on the nucleic acid to chelates with Fe +2 .
  • a nucleic acid with a group of formula -OPO 2 (NH)L 5 NH 2 bonded to the 5'-carbon of the 5'-terminal nucleotide will react with excess EDTA or DTPA, either free or complexed with a metal ion such as Eu +3 , Tb +3 or Sm +3 , in the presence of excess (relative to EDTA or DTPA) water soluble carbodiimide coupling agent, such as
  • another method of the invention for making a probe of the invention is to react a nucleic acid, with a sequence of the probe and with a group of formula -OPO 2 (NH)L 5 NH 2 , wherein L 5 is alkyl of 2 to 20 carbon atoms (preferably n-alkyl of 2 to 8 carbon atoms) bonded to the 5'-terminal carbon, with EDTA, optionally complexed with Eu +3 , Tb +3 or Sm +3 , or DTPA, optionally (and preferably) complexed with Eu +3 , Tb +3 or Sm +3 , in aqueous solution buffered to about pH 6 in the presence of a water soluble carbodiimide coupling agent.
  • L 5 is alkyl of 2 to 20 carbon atoms (preferably n-alkyl of 2 to 8 carbon atoms) bonded to the 5'-terminal carbon
  • EDTA optionally complexed with Eu +3 , Tb +3 or Sm +3
  • the preferred reactant is DTPA complexed with Eu +3 ,Tb +3 or Sm +3 .
  • the resulting probe can be purified by standard techniques, e.g., chromatographically. If the probe was made with EDTA, or DTPA, that was complexed with lanthanide ion, the probe is preferably isolated chromatographically employing 0.1 M to 0.5 M sodium citrate, pH 6.5 to 7, as the eluant.
  • probes made by reacting PITCP-EDTA or PICP-EDTA with polynucleotide with -OPO 2 (NH)L 5 NH 2 bonded to 5'-terminal carbon if the probe to be made in this process is complexed with Eu +3 ,Tb +3 or Sm +3 , but the EDTA or DTPA reactant is not, the probe of the invention, with EDTA or DTPA uncomplexed with lanthanide ion linked to the 5'-terminal carbon, is subjected to the standard probe chelation process.
  • Still another method of the invention for making a probe of the invention comprises providing a nucleic acid, with the sequence of the probe and with an amino group, of formula -NH 2 , bonded to the 5'-carbon of the 5'-terminal nucleotide, and reacting said nucleic acid with EDTA anhydride or DTPA anhydride at a pH between 6.0 and 8.0.
  • the reaction is carried out with a large molar excess of the anhydride (e.g., 10-10,000-fold over oligonucleotide concentration with reaction volume being adjusted such that the anhydride is at a concentration of 10 mg/ml) and is carried out for about 10 minutes to about 2 hours at room temperature.
  • a typical pH is 7.0, maintained with 0.1 M HEPES.
  • the product probe of the invention is separated from reactants chromatographically, as by HLPC. If the desired probe is complexed with Eu +3 , Tb +3 or Sm +3 , the probe with EDTAyl or DTPAyl bound through an amide linkage to the 5'-carbon of the 5'-terminal nucleotide is treated by the standard probe chelation process.
  • a polynucleotide with the sequence of a probe and with -NH 2 bonded to the 5'-terminal carbon can also be reacted, in the same way as polynucleotide with a group of formula -OPO 2 (NH)L 5 NH 2 bonded to the
  • yet another method of the invention to make probe of the invention comprises providing a nucleic acid with the sequence of the probe and with -NH 2 bonded to the
  • DTPA complexed with lanthanide III ion is the preferred reactant.
  • a polynucleotide with the sequence of a probe is the preferred reactant.
  • probe precursor or probe precursor, if subsequent modification to make probe entails addition of nucleotides
  • probe precursor can be prepared by any of several, well known, stepwise solid-phase techniques, such as that of Matteucci and Caruthers, supra, and Beaucage and Caruthers, supra, based on phosphoramidite chemistry, followed by HPLC isolation of the desired nucleic acid.
  • the synthesis can advantageously be carried out with an automated synthesizer, such as the Model 380A of Applied Biosystems, Inc.
  • Significant quantitites of pure, single-stranded polynucleotides of defined sequence up to about 100 nucleotides in length can be prepared by automated, stepwise, solid-phase techniques followed by HPLC purification.
  • the polynucleotides obtained from the automated synthesizer will have hydroxyl group bonded to the 3'-terminal carbon and, consequently, will be suitable as precursors of probes of the invention made by TdT-catalyzed strand extensions or, if the 3'-terminal nucleotide is a ribonucleotide, T4 RNA ligase-catalyzed ligations as described above.
  • a single-stranded DNA with sequence of probe can also be prepared by cloning into the RF-DNA of a filamentous bacteriophage, such as one of the M13 series (e.g., Ml3mpl8 or M13mp19), a double-stranded DNA which comprises a probing sequence desired for the probe, and then isolating the single-stranded circular DNA genome from phage produced by host bacteria (e.g., E. coli JM103 in the case of phage of the M13 series) transformed with the RF-DNA which includes the double-stranded DNA with probing sequence.
  • a filamentous bacteriophage such as one of the M13 series (e.g., Ml3mpl8 or M13mp19)
  • a double-stranded DNA which comprises a probing sequence desired for the probe
  • the single-stranded phage DNA can be randomly cleaved, as by sonication or with DNAse I (e.g., from bovine pancreas), to a convenient average size, preferably larger than the probing sequence, to provide DNA, with sequence of probe and with 5'-terminal or 3'-terminal phosphate groups, which can be employed, as described above, to make probe of the invention. If cleavage is with DNAse I, only the 5'-terminal nucleotide will be phosphorylated.
  • DNAse I e.g., from bovine pancreas
  • Phage DNA fragments with the 3'-carbon of the 3'-terminal nucleotide hydroxylated can be employed as described above, as precursors to make a probe of the invention enzymatically with TdT or, after addition of a 3'-terminal, 3'-hydroxylated ribonucleotide using TdT, T4 RNA ligase.
  • a double-stranded DNA which comprises a suitable sequence (e.g., a probing sequence for a target DNA or RNA), can be employed as a source of single-stranded DNA with sequence of a probe of the invention (or a precursor thereof), for modification by methods described above to make probe of the invention.
  • a suitable sequence e.g., a probing sequence for a target DNA or RNA
  • Such double-stranded DNA can also be used as a template for making a DNA or RNA probe of the invention (or precursor thereof) enzymatically, with DNA-dependent DNA polymerase, DNA-dependent RNA polymerase or TdT, as described above.
  • the above-described nick-translation method can be applied, using the double-stranded DNA as template, to make probe of the invention (or precursor thereof) (actually a mixture of probes or precursors, due to random cleavage of the double-stranded DNA template by the DNAse I).
  • a double-stranded DNA which comprises a desired sequence can be prepared by solid-phase, stepwise synthesis of each of the strands, followed by combining them in a solution for annealing into double-stranded form.
  • a double-stranded DNA which comprises a sequence, such as a probing sequence can be cloned in a suitable cloning vector (e.g., plasmid ⁇ BR322), and the cloned vector itself can be employed as DNA with sequence of probe or a portion of the vector can be excised, as by digestion of the vector with a suitable restriction endonuclease, and purified, as by agarose gel electrophoresis or any other technique suitable for separating DNAs on the basis of size, and used as DNA with sequence of probe or as a precursor of such DNA.
  • a suitable cloning vector e.g., plasmid ⁇ BR322
  • the cloned vector itself can be employed as DNA with sequence of probe or a portion of the vector can be excised, as by digestion of the vector with a suitable restriction endonuclease, and purified, as by agarose gel electrophoresis or any other technique suitable for separating DNAs on the basis of
  • probes of the invention are employed in nucleic acid hybridization assays of samples for the presence of target DNA or RNA, and, consequently, the biological entity uniquely associated with the target DNA or RNA in samples being tested.
  • the probes of the invention are used in such hyridization assays, employing standard techniques for hybridizing probe nucleic acid to target nucleic acid, as follows:
  • nucleic acid is isolated from a sample to be assayed, and is affixed in single-stranded form, to a solid or macroporous support. This procedure is carried out so that a substantial fraction (preferably most) of the target sequence for probe on the target DNA or RNA that might be present in the sample remains intact.
  • solid support and methods of affixing sample nucleic acid thereto, can be employed.
  • nitrocellulose paper can be used. See, e.g., Grunstein and Hogness, supra; Meinkoth and Wahl, supra.
  • the nucleic acid from samples can be affixed covalently by known methods directly to solid beads, such as beads of fine-grained cellulose or Sephadex TM, or "beads" of macroporous materials such as agarose (e.g., Sepharose TM or Sephacryl TM , such as Sephacryl S-500) See, e.g., Bunemann et al., Nucl.
  • solid beads such as beads of fine-grained cellulose or Sephadex TM, or "beads" of macroporous materials such as agarose (e.g., Sepharose TM or Sephacryl TM , such as Sephacryl S-500) See, e.g., Bunemann et al., Nucl.
  • a solid or macroporous support which has bound to it a first nucleic acid, said first nucleic acid including a probing segment with a sequence that is complementary to the sequence of a first target segment in target nucleic acid. After binding the first nucleic acid to the solid support, and then pre-hybridizing the support, hybridization is carried out with single-stranded nucleic acid of the sample.
  • target nucleic acid in the sample if any, becomes affixed to the solid support by base-pairing between the first target segment and the probing segment of said first nucleic acid bound to the support.
  • a second target segment of target nucleic acid, that does not overlap the first target segment, is the target segment for probe of the invention.
  • Example VIII a macroporous-support-first nucleic acid system, and methodology for making and using same, are described.
  • the support is pre-hybridized in order to substantially eliminate sites on the support for non-specific binding by probe nucleic acid.
  • this pre-hybridization step will have already taken place prior to hybridization between nucleic acid from the sample and the first nucleic acid bound to the support.
  • pre-hybridization of support is not needed after nucleic acid from the sample is affixed; but, preferably, in place of this prehybridization, the support will be washed once or twice in a wash procedure (substantially the same as the post-hybridization, high stringency, wash procedure described below) to eliminate from the support nucleic acid from sample that has not stably hybridized to the first nucleic acid bound to the support.
  • the support is exposed to a hybridization solution which contains probe of the invention at a molar concentration 10 1 -10 12 times, typically 10 3 to 10 6 times, that of target nucleic acid expected to be on the support, if the sample being analyzed included target nucleic acid.
  • the hybridization is continued for a time period sufficient for formation of duplex between probe and at least a portion (preferably most) of any target nucleic acid segment on the support.
  • unduplexed or partially duplexed probe is removed from the support by a series of post-hybridization washes, usually 1 or 2, under stringency conditions that ensure that only probe that is stably duplexed to target segment remains in the system and that probe involved in non-homologous heteroduplexes (with nucleic acid segments other than target segment of the probe) is removed from the system.
  • nucleic acid hybridization art will understand how to determine readily conditions for attachment of sample nucleic acid to solid or macroporous support, pre-hybridization of the support, and hybridization (s) and post-hybridization washes to ensure the specificity of, and achieve acceptable sensitivity for, a particular probe of the invention for a particular target nucleic acid segment in samples to be assayed with the probe. See, e.g., Meinkoth and Wahl (1984), supra.
  • Probe employed in the hybridization solution is preferably complexed, through EDTAyl, DTPAyl or p-EDTA-phenyl group (or groups) chemically linked to it, to Eu +3 , Tb +3 or Sm +3 , most preferably Eu +3 .
  • probe present on the support reflecting the presence of target DNA or RNA of the probe in the sample being assayed and the presence in the material from which the sample was obtained of the biological entity associated with said target DNA or
  • RNA is detected by excitation of fluorescence from the Eu +3 , Tb +3 or Sm +3 complexed with the probe and observation of the resulting fluorescence (i.e., fluorescence emission).
  • fluorescence emission i.e., fluorescence emission
  • sensitivity of a probe involving such a chelate and detected by fluorescence is relatively low and not amenable to enhancement by time-resolved fluorometry. Nonetheless, in assays where a probe of low sensitivity is acceptable, fluorescence can be measured directly from the support with probe bound to chelates of Eu +3 , Tb +3 or Sm +
  • DTPAyl or p-EDTA-phenyl group and water molecules are complexed with the lanthanide ion. Because the phenyl group enhances the fluorescence emission of the lanthanide ion, p-EDTA-phenyl is the preferred chelating agent-tag moiety in probes to be detected by fluorescence directly from the tag moiety/water chelate of the Eu +3 , Tb +3 or Sm +3 bound to probe.
  • a hybridization assay of a sample will be conducted in parallel with a hybridization assay of a negative control, which is a sample similar to the test sample but known to be free of target nucleic acid of probe employed in the hybridization assay, and perferably also a hybridization assay of a positive control, which is a sample similar to the test sample but known to include target nucleic acid of the probe used in the hybridization assay.
  • the assays of test sample, negative control and positive control will be run with the same reagents and procedures and at the same time. Then signal (fluorescence emission) from the sample and controls will be compared. A positive signal from positive control establishes that the assay procedures are operative.
  • one or more positive controls which include known quantities of target nucleic acid
  • comparison of fluorescence intensity from a test sample with fluorescense intensity from the negative and positive controls can be used to estimate the amount of target nucleic acid in the test sample and the titer of the associated biological entity in the material from which the test sample was prepared.
  • the preferred method for detecting probe is to proceed as follows:
  • the support with probe-lanthanide ion complex bound (if target nucleic acid of probe was in the sample being assayed), is incubated with an
  • fluorescence of the resulting solution (which will include lanthanide ion chelates in micelles if probe-lanthanide ion complex was bound to the support) is measured directly with excitation and observation of emission at wavelengths characteristic of the lanthanide ion involved.
  • the preferred lanthanide ion is Eu +3 .
  • time-resolved fluorometry is employed, using any of numerous devices for measurement of time-resolved fluorescence that are commercially available.
  • a typical enhancement solution will be an aqueous solution, will have a pH between 2.8 and 3.5 maintained with a suitable buffer (e.g., phthalate-HCl), typically at about 0.1 M concentration, will include aoout 0.1% (v/v) to about 0.5% (v/v) of a non-ionic detergent, such as Triton X-100 or a Tween (e.g., Tween-20 or Tween-80), suitable for forming micelles capable of sequestering ⁇ -diketone/Lewis base chelates of lanthanide ion from water, will include between about 10 uM and 100 uM of a ⁇ -diketone, and will include between about 10 uM and about 100 uM of a Lewis base.
  • a suitable buffer e.g., phthalate-HCl
  • a suitable buffer e.g., phthalate-HCl
  • a non-ionic detergent such as Triton X-100 or a Twe
  • the ⁇ -diketone employed in the enhancement solution is of formula R 20 (CO)CH 2 (CO)CF 3 , wherein R 20 is 2-naphthyl, 1-naphthyl, 4-fluorophenyl, 4-methoxyphenyl, or phenyl.
  • R 20 is 2-naphthyl, 1-naphthyl, 4-fluorophenyl, 4-methoxyphenyl, or phenyl.
  • the most preferred of the ⁇ -diketones is 2-naphthoyltrifluoroacetone.
  • the Lewis base employed in the enhancement solution is a synergistic (sometimes referred to in the art as "synergic") Lewis base selected from O-phenanthroline, triphenylphospine oxide, or a trialkylphosphine oxide, wherein the alkyl groups are the same or different and are each of 1 to 10 carbon atoms.
  • the most preferred of the Lewis bases is TOPO (tri-n-octylphosphine oxide).
  • a preferred enhancement solution consists of 0.1 M phthalate-HCl buffer, pH 3.2; 20 uM 2-naphthoyltrifluoroacetone, 50 uM TOPO and 0.1% (v/v) Triton X-100.
  • the enhancement solution is incubated with probe on the support at room temperature for 1 second to 24 hours, preferably about 1 minute, prior to measurement of fluorescence.
  • the enhancement solution serves to increase the fluorescence of the lanthanide ion, and thereby the sensitivity of probes of the invention, by a multistep process:
  • the buffer is of a pH near, or lower than, the pK of the carboxyl groups on the polyaminocarboxylate tag moiety-chelator linked to probe (i.e., pH 2.5-4), the tag moiety-chelator is protonated and, thereby, its dissociation constant for lanthanide ion substantially increased, resulting in release of the ion.
  • the Lewis base may also be a ligand in chelates with the lanthanide ion and increase fluorescence intensity from the ion; but, more significantly, the Lewis base interacts with ⁇ -diketone ligand in such chelates to deprotonate the ⁇ -diketone and thereby enhance fluorescence from the chelates due to the increased delocalization of charge when the ⁇ -diketone is in the anionic form.
  • the detergent forms micelles in which the diketone-lanthanide ion chelates cluster and become effectively shielded from water. Because water quenches fluorescence from lanthanide ion, the clustering in micelles arising from presence of the detergent further enhances fluorescence intensity and also enhances fluorescence lifetime from the lanthanide ion chelates. Enhanced fluorescence lifetime makes possible the use of time-resolved fluorometry to distinguish fluorescence from lanthanide ion from short-lived background fluorescence (e.g., from non-target nucleic acid and support material to which nucleic acid is affixed) and thereby enhance sensitivity of probes of the invention.
  • short-lived background fluorescence e.g., from non-target nucleic acid and support material to which nucleic acid is affixed
  • fluorescence excitation is at about 340 nm and fluorescence emission is observed at about 613 nm.
  • compounds and groups involved in the instant specification e.g., phosphate, EDTA, amino
  • phosphate, EDTA, amino have a number of forms, particularly variably protonated forms, in equilibrium with each other.
  • representation herein of one form of a compound or group is intended to include all forms thereof that are in equilibrium with each other.
  • uM means micromolar
  • ul means microliter
  • ug means microgram
  • a 29 base-pair segment of the hepatitis B virus genome has been identified, each strand of which, when employed as DNA with sequence of a probe, provide probes of surprising sensitivity and specificity in hybridization assays for diagnosis of hepatitis B infection.
  • the same is the case for the two 29 base RNA's with the RNA sequences corresponding to the sequences of the two DNA segments.
  • the 29 base-pair segment of the viral genome is:
  • RNA segments In the RNA segments, all of the nucleotides are ribonucleotides and T's in the DNA sequence are replaced by U's in RNA sequences.
  • nucleic acid probes with these four sequences.
  • the probes can be labeled for detection by any tag, including radioactive or chemical, in accordance with labels and labeling methods of the present invention or otherwise.
  • the 29-base nucleic acid segments can be made in large quantities, in highly pure form, by phosphormidite chemistry carried out on an automated synthesizer, followed by chromatographic purification, as illustrated in Example III.
  • various derivatives of the four segments which at derivatized at the 5'-terminal or 3'-terminal carbons and are intermediates in making probes, including derivatives with the combination of terminal labels indicated as follows:
  • 5'-AACCAACAAGAAGATGAGGCATAGCAGCA-3' was prepared on an Applied Biosystems Synthesizer, Model No. 380A (Applied Biosystems, Inc., Foster City, California, U.S.A.) using phosphoramidite chemistry of Matteucci and Caruthers (J. Am. Chem. Soc. 103,
  • the DTPA adduct of the hexylenediamine- derivatized polynucleotide was then prepared as described by Chu and Orgel, Proc. Nat. Acad. Sci.
  • the resulting pellet was taken up in 200 ul of io mM EuCl 3 solution containing 1 mM phathalate, pH 3.0. After 5 min., the pH was adjusted to 6-7 with NaOH and the mixture was frozen and stored at -20°C until use.
  • the pellet is taken up in 200 ul of 0.1 M sodium citrate buffer, pH 6.8, and to this solution, cooled on ice, is added 200 ul of 0.2 M HCl containing 0.2 mM EuCl 3 .
  • the pH of the resulting solution is adjusted to 3.2 with aqueous NaOH or HCl, as necessary, and the solution is incubated on ice for 15 minutes. After the 15 minutes, the pH of the solution is adjusted to 7 with 1 M NaOH, and the resulting solution is stored at -20°C until use.
  • EuCl 3 in 0.01 N HCl is prepared in the presence of 1 mM of DTPA.
  • the DTPA chelate of europium forms.
  • 200 ul of the resulting solution is added to 200 ng of ethylene diamine-derivatized oligonucleotide, prepared as described above for the hexylenediamine adduct but using ethylene diamine in place of hexylenediamine, in 150 ul of 0.1 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide.
  • the mixture is allowed to react at pH 6 at room temperature for 24 hours and the desired product is isolated by ethanol-precipitation.
  • 1-(p-amino-phenyl)EDTA is prepared as described by Sundberg, et al., J. Med. Chem., 17 1304-1307 (1974). Then, following Hemmila et al., supra, 10 ml of chloroform is added to the solution of 1-(p-amino-phenyl) EDTA and the mixture is treated with 25 mg of thiophosgene. After rapid stirring for 30 minutes, the aqueous layer is separated and washed three times with chloroform. 1-(p-isothiocyanato-phenyl)- EDTA is isolated from the dried aqueous layer.
  • the 1-(p-diazo-phenyl) EDTA (PDP-EDTA) is freshly prepared, following the procedure of Sundberg et al., supra, by treating
  • 1-(p-anino-phenyl)EDTA at about 0.2 M concentration in H 2 O, prepared as described above, with NaNO 2 /HCl, destroying excess NaNO 2 by addition of urea, and finally, diluting by addition of H 2 O to a final volume about 60 to about 70 times that of the solution of 1- (p-amino-phenyl) EDTA used as staring material.
  • the PITCP-EDTA and PDP-EDTA are chelated with Eu as follows: To 10 ml of a 3 mM solution of the PITCP-EDTA in 0.1 M HCl or the solution of PDP-EDTA prepared as just described is added with stirring 11.5 mg EuCl 3 .6H 2 O. Following the addition, the pH is brought to 7 by the addition of solid NaHCO 3 . The resulting solution is centrifuged to pellet excess europium, which precipitates about pH 6.5, and the supernatant, which is a solution of the desired chelate, is saved.
  • a solution prepared as in Example IV, that is about 3 mM in the PDP-EDTA chelate, is added 1 ml of a solution of 10 ug/ml of DNA, isolated from M13mpl8 phage, and 0.4 M borate buffer, pH 8. After stirring the resulting solution for 4 hours at 4°C, the labeled probe is purified by gel permeation chromatography on Sephadex G-50 using either 0.2 M sodium citrate, pH 6.8, or a solution of 0.01 M Tris-HCl (pH 7.0), 20 uM DTPA, and 50 uM CaCl 2 as eluant.
  • plasmid pUC19 1 ug of plasmid pUC19 (purchased from Bethesda Research Laboratories, Gaithersburg, Maryland, U.S.A., Catalog No. 5364SA) is taken up in 5 ul of 0.5 M Tris-HCl (pH 7.2), 0.1 M MgSO 4 , 1 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin. To this is added 1 nmole of the unlabeled 2'-deoxynucleoside-5'- triphosphates (dATP, dGTP, dCTP) and also 100 pmole of the DTPA-chelate of 5-allylamine-2'-deoxyuridine-5'- triphosphate prepared as follows:
  • nucleic acids comprising DTPA-chelate-5-allylamine-2'-deoxyuridines are then separated from nucleoside-5'-triphosphates and nucleoside-5'-triphosphate 5-allylamine analog and purified by chromatography over Sephadex G-50 using 0.01 M Tris (pH 7.4) as eluant.
  • the DTPA-derivatized nucleic acid is complexed with Eu +3 as follows: 200 ng of the nucleic acid is dissolved in 100 ul of a 0.1 M sodium citrate solution, pH 6.7, the solution is cooled on ice and is combined with 100 ul of a 0.2 M HCl solution with 0.1 uM
  • the pH of the resulting solution is adjusted to pH 3.2 by addition of NaOH or HCl as necessary and is then incubated on ice for 15 minutes. The pH of the solution is then raised to 6.7 by addition of 1 M NaOH.
  • the nucleic acid-Eu +3 chelate is isolated by gel permeation chromatography on Sephadex G-50 using a solution of 0.2 M sodium citrate (pH 6.8) as eluant.
  • Example VI The nick-translating procedure of Example VI is followed, except that 100 pmole of 5-allylamine-2'- deoxyuridine-5'-triphosphate is used in place of the DTPA-chelate thereof.
  • agarose beads Sephacryl S-500 macroporous support, purchased from Pharmacia, Inc., Piscataway, N. J., U.S.A.
  • the resulting suspension was filtered and then washed five times, each with a volume of cold distilled water equal to the volume of "gel” remaining on the filter, and, finally, once with the same volume of cold, 10 mM potassium phosphate buffer pH 8.
  • the "gel” was immediately transferred to a flask, to which was added quickly 6-aminocaproic acid (NH 2 (CH 2 ) 5 CO 2 H) (0.8 g per gram of "gel") and enough 10 mM potassium phosphate buffer (pH 8) to bring the volume to 8 ml per gram of "gel".
  • the resulting mixture was stirred at room temperature for 12 to 24 hours.
  • Purified complementary oligonucleotide was 5'-phosphorylated with ATP and T4 polynucleotide kinase by a standard technique.
  • the kinased nucleotide (25 ug/ml of kinase reaction solution) was then purified by adding to 0.3 ml of the solution 0.04 ml of 8 M LiCl solution and 0.9 ml absolute ethanol, freezing the resulting solution on dry ice, ⁇ entrifuging at room temperature for 10-15 minutes to form a pellet, and then withdrawing and discarding supernatant with a pulled pipette.
  • the pellet (approximately 7 ug) of the purified, kinased oligonucleotide was then dissolved in 300 ul of 0.25 M ethylenediamine ("EDA”), 0.1 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (“CDI”) and 0.1 M methylimidazole (“Melm”), pH 6.0, and allowed to react for 16 hours at 23°C.
  • EDA ethylenediamine
  • CDI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • Melm 0.1 M methylimidazole
  • EDA-derivatized oligonucleotide was then pelleted, after being mixed with LiCl and ethanol and frozen, as described above for the kinased oligonucleotide. Then, to remove any contaminating EDA, the derivatized oligonucleotide was twice taken up in 0.1 M MES buffer, pH 6, and pelleted, with LiCl/ethanol and freezing, as above. The final pellet (approximately 6 ug) was taken up into 300 ul of 0.1 M MES buffer, pH 6.0.
  • Sephacryl-500 "gel” i.e., macroporous support
  • 50 mg of support was taken from storage. washed with 0.1 M MES, and then taken up in 0.55 ml of 0.1 M CDI and 0.1 M MES buffer, pH 6, in a 1.8 ml Nunc tube.
  • 25 ul of solution of the EDA-derivatized complementary oligonucleotide (approximately 20 ng/ul) in 0.1 M MES buffer, pH 6.
  • the tube was then put on a Sepco tube rotator for stirring for 16-20 hours at room temperature.
  • the support was then pelleted by centrifugation, and then washed three times, each time by being shaken with 1.5 ml of 0.01 M NaOH, pelleted by centrifugation, and having supernatant removed by pipette.
  • the support after the final wash, was suspended until use in 10 mM Tris-HCl, 1 mM EDTA, pH 7.4.
  • the non-complementary oligonucleotide was EDA-derivatized and bound to aminohexanoic acid-derivatized Sephacryl S-500 beads by the same procedure as the complementary oligonucleotide and was bound to the same extent, approximately 0.7 pmole/mg. Hybridizations were then carried out between each of the doubly labeled polynucleotide of Example III
  • a hybridization solution was prepared by combining 750 ul of this SSC/SDS/Dextran sulfate solution with 30 mg of Sephacryl beads with oligonucleotide bound (20 pmole oligonucleotide) and 50 fmole of labeled oligonucleotide.
  • the hybridization solution was incubated for 90 minutes at 23°C.
  • the Sephacryl beads were pelleted and washed three times with 2X SSC at 23°C.
  • the quantity of labeled oligonucleotide bound to the beads was determined by measuring radioactive decay of 32 P.
  • employing a lanthanide III chelate tag to label a nucleic acid probe does not interfere with the specificity of the probe and does not interfere significantly, if at all, with the hybridization efficiency of the probe.
  • 2-Napthoyltrifluoroacetone was prepared by a modification of the method of Reid and Calvin (J. Amer. Chem. Soc 72, 2948-2949 (1950)), as follows: To 10.5 mmoles of sodium methoxide was added 20 ml of dry benzene under a nitrogen atmosphere. 10 mmoles of S-ethylthiotrifluoroacetate was added followed by
  • the fluorescence enhancement solution was prepared according to the method of Hemmila et al.. Anal. Biochem., 137, 335-343 (1984).
  • the buffer was composed of 0.1 M phthalate (pH 3.2) containing 15 uM 2-napthoyltrifluoroacetone, 50 uM tri-n-octylphosphine oxide, and 0.1% (v/v) Triton X-100.
  • Example VIII After 5 minutes incubation, the samples were illuminated with an ordinary ultraviolet lamp and visually inspected. The sample with doubly-labeled probe hybridized to complementary oligonucleotide was dark red. The sample with doubly-labeled probe hybridized to non-complementary oligonucleotide was faintly red. The other two samples remained clear. EXAMPLE XI
  • the phenyl azide-derivatized DTPA of Example XI is employed to illustrate the use of phenyl azide-derivatized DTPAs and EDTAs of the invention to label nucleic acids non-specifically with lanthanide III ion.
  • a stock solution at 1 mg/ml in water was prepared with the phenyl azide-derivatized DTPA of formula (NCH 3 )(CH 2 ) 3 NH(DTPAyl), prepared as in Example XI.
  • the solution was prepared in the dark and stored in the dark at -20°C.
  • the phenyl azide-derivatized compound is chelated in the dark with Eu as follows: To 5 ml of the approximately 1.5 mM stock solution is added 0.5 ml of 1 M HCl and then, with stirring, 2.9 mg of

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DE3806617A1 (de) * 1988-03-02 1989-09-14 Behringwerke Ag Erzeugung hochexprimierender, rekombinanter, eukaryotischer zellen
US4962045A (en) * 1988-05-02 1990-10-09 The Perkin-Elmer Corporation Time-resolved fluorimetric detection of lanthanide labeled nucleotides
SE8802574D0 (sv) * 1988-07-08 1988-07-08 Wallac Oy Oligonucleotide hybridization probes and means for the synthesis of the most preferred probes
CN1055925C (zh) * 1995-06-07 2000-08-30 武汉大学 均相合成氨羧类螯合剂内酸酐的方法
DE10105014C2 (de) * 2001-01-26 2003-03-27 Schering Ag Neues Verfahren zur Herstellung von Monoamiden der DTPA
US6677483B2 (en) 2001-01-26 2004-01-13 Schering Ag Process for the production of monoamides of DTPA
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