WO1993019206A1 - Synthese d'acides nucleiques marques par fluorescence - Google Patents

Synthese d'acides nucleiques marques par fluorescence Download PDF

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WO1993019206A1
WO1993019206A1 PCT/US1993/002422 US9302422W WO9319206A1 WO 1993019206 A1 WO1993019206 A1 WO 1993019206A1 US 9302422 W US9302422 W US 9302422W WO 9319206 A1 WO9319206 A1 WO 9319206A1
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dutp
dna
dctp
enzyme
rho
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PCT/US1993/002422
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John D. Harding
Gulilat Gebeyehu
Roger Laskin
Alberto Haces
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Life Technologies, Inc.
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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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the field of the invention is enzyme-catalyzed DNA synthesis particularly synthesis of fluorescence-labelled DNA. More particularly, the invention relates to synthesis of fluorescence-labelled nucleic acids having sequence complementary to a specified nucleic acid template.
  • Techniques for labelling nucleic acids with a fluorescence marker can be divided into two categories: post-synthetic chemical or enzymatic modification, and incorporation of fluorescence-labelled precursors during synthesis.
  • the former category see Dirks, R.W. et al. (1991) Exptl. Cell Res. 194:310-315; Lichter, P. et al. (1988) Human Genetics 80: 224-234; and Lichter, P. et al. (1990) Proc. Natl. Acad. Sci. USA 87:6634-6638.
  • the present invention belongs in the latter category.
  • a typical enzyme-catalyzed DNA synthesis reaction employs a template DNA or RNA, a primer oligonucleotide having a sequence complementary to a segment of the template DNA, an enzyme catalyst and four deoxynucleotide (dNTP) precursors, dATP, dGTP, dCTP and dTTP or, alternatively, dUTP.
  • dNTP deoxynucleotide
  • Various enzyme catalysts are known in the art, such as E. coli DNA polymerase, T7 DNA polymerase, Klenow fragment of DNA polymerase, Tag DNA polymerase, reverse transcriptase and the like.
  • the enzymes can be characterized by the degree to which an enzyme molecule remains continuously associated with the same growing strand of nascent DNA.
  • processing refers to the number of nucleotides that a DNA polymerase incorporates during DNA synthesis before dissociating from the template-primer complex.
  • a “processive” enzyme incorporates more than one nucleotide before dissociating and a non-processive (or distributive) polymerase dissociates from the template-primer after every addition of a new nucleotide.
  • the concepts of "processivity” and “high processivity” are discussed in A. Kornberg and T. Baker, DNA Replication (2nd Ed., W.H. Freeman and Co., N.Y., 1992) p. 494.
  • RNA polymerases include bacterial RNA polymerases, eucaryotic RNA polymerases I, II and III, and RNA replicases, for example, Qß replicase.
  • Fluorescence-labelling has been accomplished in the prior art by partially substituting a fluorescence-labelled NTP analog so that during synthesis an analog molecule occasionally replaces a normal NTP precursor in the sequence.
  • Various fluorescent dNTP derivatives are known in the art, having a fluorophore, such as fluorescein or rhodamine, covalently linked to the purine or pyrimidine base by a linker group.
  • Examples of prior art fluorescent dNTP derivatives are Rhodamine-6-dATP, Rhodamine-6-dCTP, Fluorescein-7-dUTP and Fluorescein-12-dUTP. The latter is available from Boehringer Mannheim Biochemica.
  • the fluorescent NTPs used in the prior art have been considered likely to interfere with synthesis due to the steric interference of the large fluorophore moiety.
  • fluorophore-substituted dNTPs have been employed as partial substituents for the normal dNTP, i.e., the synthesis reaction mixture contains less than 1.0 mole fraction of total dNTP as its fluorescent derivative.
  • a typical reaction mixture might contain a 1:3 mole ratio of Fl-12-dCTP:dCTP.
  • dNTP-X fluorescence-labelled dNTP
  • Jett et al. U.S. patent no. 4,962,037 have developed techniques for sequentially analyzing the exonuclease products of a single DNA molecule. Each deoxynucleotide labelled with a fluorophore is detected in sequence as it is released by exonuclease action, using a sensitive flow-fluorometric technique. The efficiency of the foregoing technique will be improved by providing fully fluorescent labelled DNA of greater length than available heretofore.
  • dNTP-X fluorescent dNTP
  • the invention provides a method for synthesizing nucleic acids wherein at least one of the four NTPs is completely substituted by a fluorescent-labelled ribo- or deoxyribonucleotide, r- or dNTP-X.
  • labelled DNA of at least 200 bases length can be synthesized.
  • labelled DNA of at least 500 bases length can be synthesized.
  • the method includes the use of dNTP-X compounds having the nucleotide base moiety covalently joined to the fluorophore by a linker chain of 8 to 12 atoms length. In the preferred embodiment, such linker chains are those lacking an ether linkage.
  • a processive DNA polymerase is employed.
  • single-strand DNA binding protein is provided in the reaction mixture.
  • Novel compounds suitable for use in synthesizing fully labelled fluorescent DNA are disclosed herein. These include Rho-8-dCTP, Rho-10(J)-dCTP, Fl-10(J)-dCTP, Rho-15-dCTP, Fl-15-dCTP, Rho-12-dUTP, Fl-12-dUTP, Fl-8-dATP, Rho-8-dATP, Fl-15-dATP, Res-10-dUTP, HC-6-dUTP, and Rho-12-dUTP (R).
  • linkage of the fluorophore does not modify a site on the purine or pyrimidine base that is normally involved in the hydrogen bonding interactions of base-pairing.
  • the method of the present invention has successfully produced fully single-labelled fluorescent DNA of greater than 7000 bases length.
  • the method of the invention is useful to provide fully labelled fluorescent RNA or DNA for sequence analysis, for histochemical fluorescent labelling and for micro-analytical techniques where highly fluorescent RNA or DNA of specified sequence is desired.
  • a useful kit for carrying out the method of the invention is also provided.
  • the method of the invention uses conventional enzyme-catalyzed RNA or DNA synthesis reactions in which the complementary sequence of a template nucleic acid strand is synthesized in a reaction mixture containing an RNA or DNA polymerase enzyme, four ribo or deoxy nucleotide triphosphates (r- or dNTPs) and optionally an oligonucleotide primer, depending on the specific requirements of the enzyme chosen to catalyze synthesis.
  • the template nucleic acid is usually cloned or purified DNA or RNA whose sequence is to be determined, or for which a fluorescent complement is desired.
  • the template DNA is usually in single-stranded, denatured, partially single-stranded, or partially denatured form depending on the template requirements of the polymerase employed.
  • Primer DNA is typically an oligonucleotide whose sequence is complementary to a segment of the template nucleic acid. Synthesis is considered to proceed by stepwise addition of ribo- or deoxynucleotides to the 3'-end of the primer, thereby extending the length of the primer, with concomitant release of a pyrophosphate from the precursor r- or dNTP.
  • RNA or DNA has a sequence complementary to the template sequence according to the known base-pairing relationships, A with T (or U) and G with C, of nucleic acids.
  • Enzymes which catalyze DNA synthesis are termed DNA polymerases.
  • Many DNA polymerases are known, for example T5 polymerase (Chatterjee, D., U.S. Patent 5,047,342), T7 polymerase, E. coli polymerase Klenow fragment, Taq polymerase, VentTM (New England Biolabs) polymerase and PRD1 polymerase (Savilahti, H. et al. (1991) J. Biol. Chem. 266:18737- 18744).
  • Reverse transcriptases which catalyze DNA synthesis using a RNA template, are also suitable for the present invention.
  • a wide selection of such enzymes is commercially available, including AMV-RT and M-MLV-RT (GIBCO BRL), and HIV-RT.
  • Highly processive enzymes are preferred herein.
  • a "highly processive" enzyme is herein defined as one that incorporates 50 or more nucleotides before dissociating from the template-primer complex under a given set of reaction conditions.
  • Highly processive DNA polymerases include phage T5 polymerase and derivatives thereof ("T5"), phage T7 polymerase and derivatives thereof (“T7”), phage Phi-29-type polymerases (“Phi-29”), and E. coli pol III holozyme.
  • T7 DNA polymerase (also called "T5 gene 5 protein") by itself is a DNA polymerase with low processivity. In the presence of thioredoxin cofactor, however, T7 becomes highly processive, incorporating thousands of nucleotides from a given primer without dissociation (S. Tabor et al. [1987] J. Biol. Chem. 262:16212; S. Tabor and C. Richardson, U.S. Patent 4,795,699).
  • the Phi-29-type polymerases include Phi-29, Cp-1, PRD1, Phi-15, Phi-21, PZE, PZA, Nf, M2Y, BI03, SF5, GA-1, Cp-5, Cp-7, PR4, PR5, PR722, AND L17 (Blanco et al., U.S. Patent 5,001,050).
  • E. coli pol III holozyme is a highly processive enzyme (processivity value greater than 5000), as described by A. Kornberg and T. Baker in DNA Replication (2nd Ed., W.H. Freeman and Co., NY, 1992), at pages 494-495.
  • Commercially available RNA polymerases include SP6 RNA polymerase, T3 and T7 RNA polymerases.
  • the four precursor dNTPs are dATP, dGTP, dCTP, and dTTP or, alternatively, dUTP, usually provided in approximately equimolar amounts with one another.
  • Either dTTP or dUTP can be used in a DNA polymerase-catalyzed synthesis of DNA, and for the purposes of the present invention dTTP and dUTP can, as a practical matter, be used interchangeably, as can their respective fluorescence-labelled derivatives.
  • fluorescence-labelling reactions one of the dNTPs is partially substituted by a fluorescence-labelled derivative.
  • the mole ratio of dTTP or dUTP to Fl-dUTP would typically be about 2:1.
  • the reaction product would contain Fl-dU in some sequence loci where dT or dU would be incorporated.
  • the ratio of Fl-dU to dT or dU in the product might be lower than that of the reaction mixture because reaction kinetics would favor incorporation of dT or dU over the derivative.
  • Fluorescence-labelled r- or dNTP derivatives are herein abbreviated r- or dNTP-X.
  • the r- or dNTP-X compounds of the present invention have two basic components, a fluorophore and a linker.
  • the fluorophore can be any highly fluorescent compound, including, without limitation, fluorescein, rhodamine, resorufin, and hydroxycoumarin.
  • a variety of fluorophores having different excitation and emission maxima are desirable, in applications where more than one r- or dNTP is labelled.
  • the linker is typically a chain of greater than 7 and preferably 8 to 12 atoms covalently joining the purine or pyrimidine base of the r- or dNTP to the fluorophore.
  • the linker can be shorter, preferably 5 atoms length.
  • the linker can be an aliphatic chain of C-C bonds, optionally combined with alkene groups, amide bonds, ether groups and the like.
  • r- or dNTP-X compounds lacking ether groups in the linker are preferred.
  • the linker joins the purine or pyrimidine moiety at a site or atom not involved in the hydrogen bond formation of nucleotide base pairing.
  • these sites are the 6-amino group and the 1-N of the purine ring; for guanine, the 6-OH , the 1-N and the 2-amino group; for cytosine, the 4-amino group, the 3-N and the 2-OH; and for thymine, the 4-oxo group and the 3-N.
  • dNTP-X compounds useful in the practice of the synthesis method are shown in Tables 1-4.
  • the nomenclature used herein identifies the fluorophore, the linker length (except in the case of HC-6-dUTP and Res-10-dUTP) and the nucleotide base.
  • HC-6-dUTP and Res-10-dUTP refer to the commercial names for hydroxy-coumarin-6-dUTP and resorufin-10-dUTP, respectively.
  • Abbreviations for fluorescein, rhodamine, resorufin, and hydroxy-coumarin are Fl, Rho, Res, and HC, respectively.
  • Rho-12-dUTP is dUTP joined to rhodamine by a linker chain of 12 atoms.
  • Use of a Jeffamine precursor for synthesis is indicated by including "(J)" in the abbreviated name and also indicates the presence of ether groups in the linker.
  • Use of an "(R)" immediately after the abbreviated name indicates a rigid linker as compared to the non-rigid analog; e.g., Rho-12-dUTP(R) versus Rho-12-dUTP.
  • Specific fluorescence-labelled nucleotides are designated herein as dATP-X, dGTP-X, dCTP-X and dUTP-X.
  • dNTP-X for the corresponding dNTP in a DNA synthesis reaction
  • Complete substitution for one of the r- or dNTPs means that one of the r- or dNTPs is replaced entirely by an r- or dNTP-X.
  • complete substitution of dCTP by dCTP-X means that the reaction mixture contains dATP, dGTP, dUTP and dCTP-X, but no dCTP
  • DNA synthesized in such a reaction mixture is termed herein fully single nucleotide labelled DNA. Every cytosine (C) residue in such DNA would be replaced by C-X, a fluorescence-labelled cytosine, except for that portion represented by the original primer. Fully double nucleotide labelled DNA is then DNA in which two of the four dNTPs are completely substituted for by their corresponding dNTP-X derivatives. Similarly for fully triple and quadruple nucleotide-labelled DNA.
  • Complete substitution of a dNTP-X results in inhibition of the DNA synthesis reaction.
  • the extent of inhibition varies with the polymerase used and the dNTP-X. Inhibition from 40% to 99% relative to control reactions using the unmodified dNTPs was observed. In general reactions inhibited more than 95% failed to yield fully labelled DNA of greater than 500 bases length. The extensive inhibition observed with complete substitution may have been a significant factor in directing the art away from complete substitution heretofore.
  • kits combining the main components for carrying out the invention.
  • the kit includes a DNA polymerase enzyme and an assortment of dNTPs and dNTP-X substrates.
  • a kit designed for complete substitution of one dNTP-X will contain 3 dNTPs and one dNTP-X, either separately packaged or premixed.
  • a complete set of dNTPs and dNTP-X derivatives is provided, to facilitate any desired combination of complete single, double or multiple fluorescence labelling.
  • kits optionally provide a standard template DNA and standard primer for control and calibration, however, the end user will provide the desired template and primer for the specific desired purpose.
  • N 4 -Aminohexyl dCTP (precursor of Rho-8-dCTP, see Table 5) and N 4 -Jeffamine-dCTP (precursor of Rho-10(J)-dCTP and F1-10(J)-dCTP) were prepared by the transamination method of Draper (1984) Nucl. Acids Res. 12:989-1003.
  • N 6 -Aminohexyl dATP (precursor of Fl-8-dATP and Rho-8-dATP) was prepared by the method of Gebeyehu et al. (1987) Nucl. Acids Res. 15:4513-4534. Allylamine dUTP (precursor of Fl-12-dUTP, Rho-12-dUTP and Rho-4-dUTP) was prepared according to Langer et al. (1981) Proc. Natl. Acad. Sci USA 78:6633-6637.
  • the amino-nucleoside triphosphates (10-20 ⁇ mol) were dissolved in sodium bicarbonate (0.4 M, 500 ⁇ l) or sodium borate solution (0.1 M) and treated with a 3 to 5-fold molar excess of the N-hydroxysuccinimide ester of the dye in anhydrous dimethyl formamide (500 ⁇ l). The mixture was reacted for 3 - 18 hr. at room temperature. The reaction was monitored by thin layer chromatography (silica gel; butanol: acetone: acetic acid: 5% ammonium hydroxide: water/70:50:30:30:20) and/or by HPLC.
  • the crude mixtures were diluted in water (200 - 300 ml), loaded on a 10 - 15 cm long, by 1 cm diameter column of mild anion exchange resins and eluted, sequentially, with 0.01, 0.2 and 0.5 M triethylammonium bicarbonate until the fraction containing the fluorescent dNTP was collected. After desalting of the appropriate column fraction, TLC, HPLC and capillary electrophoresis analysis was used to assess the purity and characteristic elution patterns of the desired product. The compounds were characterized by their U.V. spectra as the overlapping spectra of the starting amino modified base and the dyes. Yields of fluorescent nucleotides were 50 - 60%.
  • the fluorescent dNTPs used in these studies are shown in Table 1.
  • the number in the abbreviated name of each compound refers to the length of the linker which joins the fluorphore to the base moiety of the dNTP.
  • Fluorescent dNTPs were synthesized by standard methods.
  • the dNTPs listed as “Orange” or “Green” contain modified Rhodamine or Fluorescein dyes, respectively and are proprietary products of Imagenetics, Inc., Naperville, 111.
  • the following DNA polymerases were tested: 1) T5 DNA polymerase modified to lack 3' to 5' exonuclease activity [T5(Exo-), BRL]. 2) T7 DNA polymerase modified to lack 3' to 5 ' exonuclease activity [T7(Exo-); U.S. Biochemical]. 3) The Klenow fragment of E. coli DNA polymerase I (BRL). 4) T. aquaticus DNA polymerase (Cetus). 5) T. litoralis DNA polymerase (VentTM, New England Biolabs). 6) Phage PRD1 DNA polymerase.
  • DNA polymerases are typical of the following general types of enzymes: 1) Highly processive E. coli enzyme (T5(Exo-), T7(Exo-), Phi-29-type such as PRD-1, and E. coli pol
  • a typical reaction in which the effect, for example, of Rhodamine-8-dCTP on synthesis of DNA by T5(Exo-) DNA polymerase was determined consisted of the following: In a total volume of 15 microliters: 27 mM Tris-HCl, pH 7.5, 13 mM MgCl 2 , 33 mM NaCl, 7 mM dithiothreitol, 1 ⁇ g M13mpl9 + Strand DNA, 5 ng 23 bp primer (BRL), 100 ⁇ M each, dATP, dGTP, dTTP, 100 to 500 ⁇ M Rho-8-dCTP and 2 ⁇ Ci, 32 P-dGTP.
  • the reaction was incubated at 37°C for 1 hour and 8 ⁇ l were removed. Of this, 3 ⁇ l were TCA precipitated to test for the effect of the fluorescent dNTP on activity and the remaining 5 ⁇ l were run on an alkaline agarose gel according to the procedure of Maniatis et al. (1982) "Molecular Cloning, A Laboratory Manual", first edition. Radioactive size markers (1 KB ladder, BRL) were also run on the gel to allow comparison of the apparent molecular weight of the reaction products with the standards following autoradiography of the gel. In addition, fluorescent DNA molecules in the gel could be visualized directly by placing the gel on a U.V. light transilluminator. The fluorescence pattern parallelled the radioactivity pattern. The remainder of the reaction was incubated overnight at 37°C and aliquots assayed by TCA precipitation and alkaline agarose gel electrophoresis, as above.
  • reaction conditions for each of these experiments was as follows. All reactions were performed in a final volume of 15 microliters containing 1 ⁇ g M13mp19 + strand DNA, 5 ng 23 bp primer, 2 ⁇ Ci 32 P dGTP, and, respectively in two different reactions, 100 or 500 ⁇ M of the fluorescent dNTP, and the specific buffers, concentrations of normal dNTPs and enzyme units listed below. The same results were obtained at either concentration of the fluorescent dNTP. Reactions were performed for 1 hour and overnight at the given temperature (see below) and assayed by TCA precipitation and alkaline agarose gel electrophoresis, as described above. Other aspects of the reaction conditions were:
  • T5 (Exo-) and T7 (Exo-) DNA polymerases Normal dNTPs (100 or 200 ⁇ M; results identical), Reaction buffer (as above), Reaction temperature (37°C), Enzyme (1 unit).
  • VentTM and Vent (Exo-)TM polymerases Normal dNTPs (200 ⁇ M), Reaction buffer (10 mM KC1, 10 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl, pH 8.8, 8 mM Mg 2 (SO 4 ) 2 , 0.1% Triton X-100, 100 ⁇ g/ml bovine serum albumin), Reaction temperature (72°C), Enzyme (1 unit).
  • T5(Exo-) and T7(Exo-) polymerases were tested with combinations of fluorescent dNTPs, as shown in Table 6.
  • the overnight reactions were carried out exactly as described above except that both of the two fluorescent dNTPs were present at 100 ⁇ M and the two remaining normal dNTPs were present at 200 ⁇ M concentrations.
  • Table 6 only certain combinations of the dNTPs gave products greater than 500 bases in size.
  • Single Strand DNA Binding Protein Single Strand DNA Binding Protein.
  • the following reaction was performed. Fifteen microliter reactions, basically as described above for T5(Exo-) DNA polymerase, were performed which contained 1 unit of T7(Exo-) DNA polymerase, 0.25 or 1.0 ⁇ g of M13mp19 + strand template, respectively, 100 ⁇ M Rho-8-dCTP, 100 ⁇ M dATP, dTTP and dGTP and 6 ⁇ g of Single Strand Binding Protein (purchased from Pharmacia).
  • the Single Strand Binding Protein enhanced synthesis of radioactive, fluorescent DNA by 205% and 168%, respectively, with the two amounts of template in a one hour reaction relative to a reaction that contained Rho-8-dCTP but no Single Strand binding Protein.
  • reactions containing Single Strand Binding Protein contained fluorescent DNA of sizes 500 bp to 5 kb as compared to 500 bp to 2 kb in reactions lacking the protein. This proves that Single Strand Binding Protein can significantly enhance the synthesis of fluorescent DNA.
  • That probes suitable for in situ hybridization can be produced using direct incorporation of fluorescent nucleotides was shown by the following experiment.
  • a synthesis reaction similar to that described above, was performed using Rho-8-dCTP as the fluorescent dNTP, human Cot-1 DNA as template (Human Cot-1 DNA is total human genomic DNA greatly enriched for highly repetitive sequences and was purchased from BRL), random hexanucleotide primers (BRL) and Klenow DNA polymerase.
  • the Human Cot-1 DNA will potentially hybridize to human DNA present in rodent-human hybrid cell lines, thus allowing the identification of the particular human chromosome contained in the rodent cell.
  • the fluorescent DNA copy of the human Cot-1 DNA was hybridized to metaphase spreads prepared from the WA-17 cell line, which contains human chromosome 21 in a mouse chromosome background, by standard procedures.
  • the single copy of Human Chromosome 21 present in the cell line was readily detected by fluorescence microscopy and mouse chromosomes were not labelled by the probe.
  • Example 5 Effect of dATP Derivatives on Polymerases
  • HC-6-dUPT and “Res-10-dUTP” refer to the commercial names for Hydroxy-coumarin-6-dUTP and Resorufin-10-dUTP, respectively, and thus the numbers in the abbreviated names do not refer to the length of the linker for these compounds.
  • both bio-7-dATP and AH-dATP are better substrates than the fluorescent dATPs, and both produce larger reaction products.
  • Reactions containing bio-7-dATP produced DNA of sizes 1000-1600 bases and AH-dATP (linker only) produced DNA of sizes 500-2000 bases; reactions containing fluorescent dATPs produced fluorescent DNA of sizes less than 500 bases.
  • This proves that incorporation of biotin dATP or AH-ATP is not predictive of the incorporation of the cognate fluorescent dATPs, nor does synthesis of biotinylated DNA predict the synthesis of large fluorescent DNAs.
  • T5(Exo-) DNA polymerase is a better enzyme for incorporating biotin dATP and AH-ATP than is the Klenow polymerase, as shown by the percent activities.
  • Rho-7-dATP at a high concentration is a much better substrate for T5(Exo-) polymerase (24.6% activity) than it is for Klenow polymerase (1.9% activity).
  • Both bio-7-dATP and AH-dATP produce larger reaction products than fluorescent dATPs, which produced fluorescent DNA of sizes less than 500 bases.
  • Rho-7-dATP is a much better substrate for the T5(Exo-) polymerase as the concentration is raised from 25 to 100 ⁇ M; the activity of Fl-7-dATP, on the other hand, decreases with increasing concentration.
  • Rho-12-dUTP and Rho-12-dUTP differ in the structure of the linker, the latter compound having a more rigid linker than the former.
  • the reaction conditions were identical to those of Example 2 except that the reactions were performed overnight with one of the following polymerases: T5(Exo-), purchased from BRL; T7(Exo-), purchased from U.S. Biochemical; AMV-RT and M-MLV-RT, purchased from BRL; and HIV-RT, obtained from Dr. Steven Hughes of the National Cancer Institute.
  • the three reverse transcriptases (AMV-RT, M-MLV-RT and HIV-RT) copied DNA templates and thus were used as DNA polymerases. Reactions were assayed by TCA precipitation and alkaline agarose gel electrophoresis, as described above. The results are summarized in Table 10.
  • HC-6-dUTP is a particularly good substrate with T5(Exo-) and T7(Exo-) polymerases and promotes the synthesis of large fluorescent DNAs (some greater than 7 kb in size).
  • the rigid linker of Rho-12-dUTP (R) is particularly advantageous for T5(Exo-) DNA polymerase relative to Rho-12-dUTP.
  • the reverse transcriptases synthesize fluorescent DNAs greater than 200 bases in size using certain dNTPs, particularly HC-6-dUTP and Rho-12-dUTP. Like other polymerases, the reverse transcriptases also vary in their abilities to incorporate certain fluorescent dNTPs (see above).

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Abstract

L'invention se rapporte à un procédé de synthèse d'acides nucléiques possédant une longueur d'au moins 200 ou 500 bases, y compris des brins atteignant une longueur de plusieurs kb, dans lequel au moins un des quatre NTP est totalement substitué par un ribo ou désoxyribonucléotide marqué par fluorescence r- ou dNTP-X. Le procédé comprend l'utilisation de composés de dNTP-X dont la fraction de base de nucléotide est reliée de façon covalente au fluorogène par une chaîne de liaison d'une longueur de 8 à 12 atomes, l'utilisation d'une ADN polymérase processive, ainsi que l'utilisation d'une protéine de liaison d'ADN à monobrin dans le mélange réactionel. L'invention décrit également de nouveaux composés s'utilisant dans la synthèse de l'ADN fluorescent complètement marqué, ainsi qu'un kit servant à mettre en application ledit procédé. Le procédé décrit par l'invention est efficace dans l'obtention d'ADN ou d'ARN fluorescent totalement marqué servant à effectuer des analyses de séquences, dans le marquage fluorescent histochimique, ainsi que dans des techniques microanalytiques nécessitant un ADN ou un ARN extrêmement fluorescent et possédant une séquence spécifique.
PCT/US1993/002422 1992-03-17 1993-03-17 Synthese d'acides nucleiques marques par fluorescence WO1993019206A1 (fr)

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US85270592A 1992-03-17 1992-03-17
US07/852,705 1992-03-17

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WO1993019206A1 true WO1993019206A1 (fr) 1993-09-30

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000011220A1 (fr) * 1998-08-21 2000-03-02 Washington University Polarisation de fluorescence utilisee dans l'analyse d'acides nucleiques
EP2036897A1 (fr) * 2007-09-04 2009-03-18 Roche Diagnostics GmbH Réactif de marquage stable à base de rhodamine

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Title
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BIOTECHNIQUES, Volume 8, No. 6, issued 1990, J.J. LANZILLO, "Preparation of Digoxigenin-Labelled Probes by the Polymerase Chain Reaction", pages 620-622. *
ELECTROPHORESIS, Volume 13, issued September 1992, W. ANSORGE et al., "High-Throughput Automated DNA Sequencing Facility with Fluorescent Labels at the European Molecular Biology Laboratory", pages 616-619. *
JOURNAL OF CELLULAR BIOCHEMISTRY, Supplement 16B, issued 1992, M.L. HAMMOND et al., "Enzymatic Synthesis and Exonucleolytic Degradation of Fluorescent DNA Containing Rhodamine and Fluorescein Nucleotides", see Abstract F325. *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000011220A1 (fr) * 1998-08-21 2000-03-02 Washington University Polarisation de fluorescence utilisee dans l'analyse d'acides nucleiques
US6180408B1 (en) 1998-08-21 2001-01-30 Washington University Fluorescence polarization in nucleic acid analysis
US6440707B1 (en) 1998-08-21 2002-08-27 Washington University Fluorescence polarization in nucleic acid analysis
EP2036897A1 (fr) * 2007-09-04 2009-03-18 Roche Diagnostics GmbH Réactif de marquage stable à base de rhodamine

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