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WO1994005797A1 - Synthese in vitro de molecules d'adn - Google Patents

Synthese in vitro de molecules d'adn Download PDF

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Publication number
WO1994005797A1
WO1994005797A1 PCT/FI1993/000340 FI9300340W WO9405797A1 WO 1994005797 A1 WO1994005797 A1 WO 1994005797A1 FI 9300340 W FI9300340 W FI 9300340W WO 9405797 A1 WO9405797 A1 WO 9405797A1
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dna
enzyme
thermophilic
polymerase
polymeraεe
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Vsevolod Kiselev
Evgenii Severin
Timo Korpela
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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 present invention is directed to improvements relating to enzymatic synthesis of DNA molecules in vitro by a com ⁇ bination of thermophilic DNA poly erases and thermophilic pyrophosphatases in the same mixture.
  • the polymerases are involved in carrying out the synthesis of DNA by polymeri- zation of dNTP, while the pyrophosphatase eliminates py- rophosphate that is accumulated during this process, hereby increasing its efficiency of amplification.
  • RNA molecules are of great importance for detecting oncogene expression.
  • concentration and primary structure of RNA, coded with oncogenes can provide an opportunity to make a prognosis of cell oncotransformation.
  • Analysis of RNA molecules normally consists of two stages. In the first stage, the synthesis of copy DNA is carried out with the help of a specific oligonucleotide primer, which is complementary to the target mRNA, and reverse trans- criptase. Then the copy DNA is amplified by the PCR tech ⁇ nique for the subsequent structural analysis. During the analysis of RNA by this method two problems appear.
  • the first problem deals with a strong secondary structure of the RNA molecule, which prevents the synthesis of a full copy DNA due to premature termination. This problem can be overcome if the cDNA synthesis is performed at a high temperature, which causes melting of the secondary struc ⁇ ture region of RNA.
  • the second problem is connected with the low concentration of mRNA in the samples, which does not allow its amplification during 25-35 cycles.
  • DNA sequencing method is based on the understan ⁇ ding of the fundamental mechanism of DNA synthesis (Sanger F. et al., Proc. Natl. Acad. Sci. USA, 1977, 74 . , 5463) .
  • DNA sequencing generally involves enzymatic synthesis of a single strand of DNA from a single stranded DNA template and a primer. Usually four separate syntheses are carried out on a single stranded template, being provided along with a primer which hybridizes to the template.
  • a specific base for example, a dideoxynucleotide.
  • Enzymes currently used for this method of sequencing include a large fragment of Escherichia coli DNA-polymerase I ("Klenow" fragment) , reverse transcripta- se, Taq polymerase and a modified form of bacteriophage T7 DNA polymerase.
  • Some DNA templates have a very strong secondary struc- ture and in order to read this DNA region a high tempera ⁇ ture during the reaction should be used to melt the se ⁇ condary structure.
  • Degradation of a fragment can occur via a nucleophilic attack at the 3'- terminal internucleotide linkage by H_0 or PPi (pyrophosphate) .
  • the former reaction is catalysed by the 3' to 5'-exonucleases activity associated with many DNA polymerases, generating dNMP or ddNMP.
  • the latter reaction is pyrophosphorolysis, the reversal of polymerization, and involves generation of dNTP and ddNTP (Deutscher, M. P. and
  • PCR The PCR method is today the main approach in manipulation with nucleic acids. Most of genetic engineering research is based on the application of PCR. Actually all molecular genetic analyses in clinical practice, aimed at diagnosing human genetic disorders, are carried out with PCR. PCR is also widely used in the diagnosis of infectious diseases, especially in the cases when immunological methods cannot be used. Careful analy ⁇ is of the many publications dealing with the application of PCR for diagnostic purpose ⁇ demon ⁇ strates that under real clinical conditions it is not possible in some cases to amplify target DNA. Some authors find an explanation for this in that Taq polymerase gradually loses its activity during PCR, which decreases the sensitivity. Hence many publications are devoted to ways of enhancing the ther o ⁇ tability of recombinant DNA- polymerases by protein engineering method ⁇ or to screening new thermoresistant enzymes.
  • the present invention aims at improving the efficacy of enzymatic synthesis of DNA in vitro. It is namely our belief that the main reason for the decreased PCR ⁇ ensi- tivity is that during DNA-synthesis some product ⁇ which are able to inhibit polymerase activity are accumulated. Also the accumulation of such products must occur more rapidly during analysis of such nucleic acid samples, wherein the target DNA is present with a few copies. Under such conditions there is a po ⁇ ibility of wrong inte- raction of primers with non-complementary or partly comp ⁇ lementary DNA templates. These interactions do not lead to amplification of target DNA, but promote the accumulation of products, which inhibit DNA-polymerase activity.
  • the present invention aims at removing ⁇ uch accumu ⁇ lated products, in particular pyrophosphate formed, by using a combination of enzymes, ⁇ pecifically a thermophi ⁇ lic DNA polymera ⁇ e enzyme in combination with a thermo ⁇ philic pyropho ⁇ phata ⁇ e enzyme.
  • thermophilic enzyme ⁇ and u ⁇ e thereof a ⁇ a tool for nucleic acid ⁇ ynthe ⁇ i ⁇ in vitro, the one enzyme being able to carry out the polymerization of dNTP within the temperature interval of 56-90 °C , i.e. a thermophilic DNA-poly era ⁇ e, and the other being able to eliminate accumulated py- ropho ⁇ phate within the ⁇ ame temperature interval, i.e. a thermophilic pyropho ⁇ phatase.
  • the invention also concerns an improvement in a method for in vitro enzymatic synthesi ⁇ of nucleic acid molecules, the improvement consi ⁇ ting in that a thermophilic DNA-polyme ⁇ ra ⁇ e i ⁇ used which is able to carry out the polymerization of dNTP within the temperature interval of 56-90 °C, in combination with a thermophilic pyrophosphata ⁇ e which is able to eliminate accumulated pyropho ⁇ phate within the same temperature interval.
  • thermophilic pyropho ⁇ phata ⁇ e i.a. from Thermus thermophilus (Tth pyrophosphatase) and used it in combina ⁇ tion with polymera ⁇ e from Thermus thermophilus (Tth polymerase) for amplification.
  • Tth pyrophosphatase Thermus thermophilus
  • Tth polymerase Thermus thermophilus
  • Pyropho ⁇ phate concentration ⁇ of 0.2 to 0.5 mM can modula ⁇ te the DNA-polymera ⁇ e function and lead to premature ter ⁇ mination of the DNA synthesi ⁇ .
  • Thi ⁇ can prevent effective amplification of a long DNA fragment.
  • Experimental data confirm our finding ⁇ and demonstrate that amplification of a lambda DNA fragment 10 kiloba ⁇ e ⁇ long i ⁇ carried out more effectively in ca ⁇ e the enzyme combination of the invention wa ⁇ u ⁇ ed.
  • the modulating effect of a high concentration of py ⁇ rophosphate on the functions of DNA polymera ⁇ e can al ⁇ o lead to decreasing fidelity and be accompanied with an increa ⁇ e of error number ⁇ during the DNA ⁇ ynthesis.
  • the enzyme combination according to the invention can advantageously be used al ⁇ o for DNA sequence analysi ⁇ to read a ⁇ trong ⁇ econdary ⁇ tructure region and a ⁇ a solution of the problems related to the accumulation of pyrophosphate.
  • a ⁇ Tth polymera ⁇ e ha ⁇ rever ⁇ e tran ⁇ criptase activity the possibility of application of the enzyme combination ac ⁇ cording to the invention for direct amplification of RNA molecule ⁇ ha ⁇ al ⁇ o been inve ⁇ tigated.
  • Such approach gives an opportunity of omitting the step of copy DNA synthe ⁇ i ⁇ with the help of AMV (Avian Myelobla ⁇ to ⁇ i ⁇ Virus, a com ⁇ flashal product of Pharmacia) reverse transcripta ⁇ e.
  • thermo ⁇ table DNA polymerase for reverse tran ⁇ cription, in which ca ⁇ e pre- dominantly full-length products can be obtained.
  • cDNA can be amplified in the polymerase chain reaction with the same enzyme.
  • RT/PCR reverse trans ⁇ cription reaction and PCR amplification
  • the enzyme mixture has been used for RT/PCR with three different templates: pAWIOS RNA template from Gene AmpR PCR kit ("Cetu ⁇ ") , 16S RNA from Bacillus ⁇ ubtilis and Influenza viru ⁇ type A RNA.
  • the invention it is possible to overcome the present restriction ⁇ on PCR, and for example to increa ⁇ e the number of cycle ⁇ , ⁇ uch a ⁇ up to 40, to be performed for the detection of single molecules of nucleic acids, without experiencing the inhibitory effect of pyrophosphate.
  • Accor ⁇ ding to the invention it i ⁇ al ⁇ o possible to determine successive ⁇ ively different agent ⁇ in one and the same sample, as no pyrophosphate is accumulated in the sample.
  • Thi ⁇ i ⁇ of cour ⁇ e an advantage e.g. when there is a limited availability of sample material.
  • thermostable enzyme combination i ⁇ al ⁇ o po ⁇ sible to provide, due to the permanent intensity of bands, low background level and high precision, sequencing sy ⁇ tems based on the thermostable enzyme combination, which makes it possible to read sequences of 10 to 700 nucleotides long.
  • the system can be recommended for a wide range of templates such a ⁇ amplified DNA, big double- ⁇ tranded DNA-template ⁇ (such a ⁇ lambda) , GC-rich templates and long poly(A) tails.
  • thermophilic DNA-polymera ⁇ e from a ther ⁇ mophilic microorgani ⁇ m, ⁇ uch a ⁇ Thermus thermophilus mic- roorgani ⁇ ms.
  • thermophilic microorganisms may be used as the enzyme ⁇ ource, such a ⁇ Thermus aquaticus and Thermus ruber.
  • thermophilic DNA-polymerases are for example Tag-polymerase (Promega) or Vent-DNA polymerase (Biolab) (see also Ruttiman C. et al., DNA-polymerase from the Extremely Thermophilic Bacterium Thermus thermophilus HB-8. 1985, Eur. J. Biochem. V. 149, pp. 41-46; Glukhov, A.I. et al., Amplification of DNA Sequences of Epstein- Barr and Human Immunodeficiency Viru ⁇ u ⁇ ing DNA-polymerase from Thermus thermophilus . 1990, Mol. Cell. Probes, Vol. 4, pp.435-443; Barballeira, N.
  • a main property of these enzymes is that they do not loo ⁇ se their activity at high temperature and can be u ⁇ ed to solve secondary structure problems in the DNA sequencing analysis and in PCR.
  • the enzyme source is not critical as long as the enzymes obtained exhibit the required properties.
  • the polyme ⁇ rase enzyme has the distinctive feature that it i ⁇ able to make copy DNA u ⁇ ing an RNA template becau ⁇ e it possesses reverse transcriptase activity.
  • the ⁇ econd component of the enzyme combination i ⁇ a ther ⁇ mophilic pyropho ⁇ phata ⁇ e, i.e. inorganic pyropho ⁇ phata ⁇ e (IP) which i ⁇ able to eliminate accumulated pyrophosphate by pyrophosphoroly ⁇ is.
  • IP inorganic pyropho ⁇ phata ⁇ e
  • thermophilic pyropho ⁇ phata ⁇ e for the purpo ⁇ es of thi ⁇ invention i ⁇ preferably isolated from Thermus thermophi ⁇ lus , but can also be obtained from other thermophilic icroorganism ⁇ , ⁇ uch a ⁇ Thermus aquaticus and Thermus rujbe .
  • thermophilic polymerase and the thermophilic pyrophosphata ⁇ e are both i ⁇ olated during the same procedure from the same mic- roorgansim, such a ⁇ Thermus thermophilus .
  • the bacterial cell ⁇ are di ⁇ integrated e.g. by ult- ra ⁇ onication in a suitable buffer (e.g.
  • a suitable ion exchange column such as DEAE- Sepharose
  • the two enzymes may be eluted with ⁇ uitable NaCl gradients, such as those disclo ⁇ ed in the Examples.
  • the pyrophosphatase and the DNA-polymera ⁇ e fractions are pooled re ⁇ pectively, and thereafter purified individually, ⁇ uch as by ion exhange chromatography or by hydrophobic chromatography.
  • a wide range of pyropho ⁇ phatase activities can be used, such as e.g. from 0.04 to 0.5 U.
  • a ⁇ uitable ratio between polymerase and phosphatase activities is e.g. 20:1.
  • the polymerase activity used for one PCR reaction with a total volume of e.g. 50 ⁇ l, i ⁇ conventionally e.g. 2.5 U, a suitable pyrophosphatase activity then being 0.125 U, and the enzyme combination being added e.g. in a volume of 0.5 ⁇ l .
  • the polymerase activity unit is defined as follows.
  • One unit of DNA-polymerase activity corresponds to an amount of the enzyme which incorporates 10 n ol of dNTPs into an acid-insoluble fraction during 30 min after a 10 min in ⁇ cubation at 74 °C under the following condition ⁇ : 25 mM TAPS pH 9.3 (at 25 °C) ; 50 mM KCl, 2 mM MgCl 2 , 1 mM jS-mer- captoethanol; 200 ⁇ M each of dATP, dGTP, dTTP; 100 ⁇ M dCTP (a mixture of unlabelled and ⁇ -[ 32 P]-labelled) ; 12.5 ⁇ g of activated DNA of ⁇ almon ⁇ perm, final volume 50 ⁇ l.
  • the pyrophosphatase activity unit is the enzyme amount that produces 2 ⁇ mol of pho ⁇ phate from pyropho ⁇ phate in 1 min at +75 °C under the following condition ⁇ : 1 mM pyrophosphate, 2 mM MgCl 2 , 50 mM Tri ⁇ HC1 pH 9.0 (at + 25°C) .
  • Fig.l illu ⁇ trate ⁇ a compari ⁇ on of thermo ⁇ tability of pyropho ⁇ phata ⁇ e from Thermus thermophilus and Escherichia coli with re ⁇ pect to the number of cycle ⁇ in PCR.
  • Fig. 2 illu ⁇ trates amplification of lambda DNA in the presence of different concentrations of pyrophosphata ⁇ e under u ⁇ ual PCR condition ⁇ for amplification of 500 bp fragment of lambda DNA.
  • Fig. 3 illu ⁇ trate ⁇ the ⁇ ynthe ⁇ i ⁇ of a 10 kilobase fragment of lambda DNA and the effect of using the enzyme combina- tion.
  • Fig. 4 illustrate ⁇ incorporation of P dNTP into synt ⁇ hesized DNA in PCR with and without pyrophosphata ⁇ e from Thermus thermophilus .
  • Fig. 5 illustratesthe efficiency of amplification of a 8 kb lambda DNA sequence u ⁇ ing different enzyme combinations.
  • Fig. 6 illustrate ⁇ the efficiency of u ⁇ ing RT/PCR using CD 4 mRNA as template.
  • the debri ⁇ was precipitated by centrifugation at 10 000 g, the supernatant was applied to a column (2.6 x 40 cm) of DEAE-Sepharo ⁇ e 4B, equilibrated with buffer A.
  • the column wa ⁇ wa ⁇ hed with the ⁇ ame buffer (about 600 ml) , the protein ⁇ were eluted with 0.025 to 0.25 M NaCl gradient (the re ⁇ t of the component ⁇ a ⁇ in the buffer A) .
  • Tth polymerase was eluted with 0.09-0.15 M NaCl
  • IP was eluted with 0.13- 0.16 M NaCl.
  • IP-containing fractions were pooled, and the purifi- cation procedure was thereafter performed individually for Tth polymerase and IP.
  • the 0.13-0.16 M NaCl fractions received from the DEAE- Sepharo ⁇ e 4B column were pooled, dialyzed again ⁇ t buffer A and rechromatographed on a column (1.6x40 cm) of DEAE- Toyopearl 65 F.
  • the proteins were eluted with a 0.05-0.35 M NaCl linear gradient.
  • Tth polymerase containing frac- tion ⁇ were collected and u ⁇ ed for further purification.
  • the IP-containing fraction ⁇ were combined and salted out with (NH 4 ) 2 S0 4 , the latter being added up to 75 % saturation.
  • the sediment was dis ⁇ olved in 1.5 M (NH 4 ) 2 SO. (the rest of the component ⁇ except (NH 4 ) 2 S0.
  • Tth polymera ⁇ e Fraction ⁇ obtained from the DEAE-Sepharo ⁇ e 4B and DEAE- Toyopearl 65F were combined, dialyzed again ⁇ t a buffer containing 0.02 M K-pho ⁇ phate pH 7.0, 0.1 M NaCl, 0.1 mM EDTA, 2 mM DTT and applied to a column (1.6x20cm) of pho- sphocellulose P-ll (Whatman) , equilibrated with the same buffer. The proteins were eluted with a 0.1-0.6 M NaCl gradient in the same buffer.
  • the polymerase-containing fraction ⁇ (0.3-0.4 M NaCl) were combined and applied to a column (1.6x20 cm) of hydroxya- patite HT (BioRad, Biogel HT) , the protein ⁇ were eluted with a 0.02-0.25 M K-pho ⁇ phate gradient, pH 7.0.
  • the polymerase-containing fraction ⁇ were combined and chromatographed on a 2.6x60 cm column of Toyopearl HW-60F.
  • the active fraction ⁇ were combined, concentrated and dialyzed against a buffer containing 0.1 M NaCl, 0.02 M K- pho ⁇ phate, pH 7.0, 0.1 mM EDTA, 2mM DTT, 50 % (w/w) glycerol, and ⁇ tored at -20°C.
  • the respective yield from 50 g of bioma ⁇ s was as follows: Tth polymera ⁇ e 25000 units, inorganic pyrophosphata ⁇ e 3000 units. Example 2 .
  • thermo ⁇ tability Compari ⁇ on of the thermo ⁇ tability of pyropho ⁇ phata ⁇ e from Thermus thermophilus and Escherichia coli in the enzyme mixture.
  • Enzyme activity wa ⁇ assayed in a medium containing 50 mM Tris.HCl pH 9.0, 2 mM MgCl 2 , 1 mM pyrophosphate.
  • the unit of activity is the amount of enzyme needed for the tran ⁇ - formation of 1 mM pyropho ⁇ phate to pho ⁇ phate during 1 minute.
  • the method of pyrophosphate a ⁇ say is based on a colour reaction, which is run after the interaction of phosphate with Na olybdate. In the reaction phosphomolyb- date i ⁇ formed followed by reduction with ⁇ tannic chloride.
  • the enzymes were incubated in PCR buffer (67 ir ⁇ Tris.HCl, pH 8.8; 16 mM (NH 4 ) 2 S0 4 ; 1.5 mM MgCl 2 ; 0.01 % Tween-20) .
  • the PCR cycle consisted of three stages:
  • the samples contained PCR buffer, 0.2 M of each dNTP, 0.3 mM of each primer, 2 units of Tth polymerase, 0.1 ng of lambda DNA.
  • One of the sample ⁇ contained 0.2 units of Tth pyrophosphatase. Amplification was performed as follows:
  • the condition ⁇ of the experiment are the same a ⁇ in the previou ⁇ example.
  • the ddN/dN mixture was prepared in 25 M Tri ⁇ .HCl and 7 mM MgCl 2 and contained the following:
  • the sequencing buffer used in step 1 is made up of ' 25 mM TrisHCl pH 8.8; 1 mM MgCl 2 , 0.1% Nonidet P40 and 0.1% Twe- en-20.
  • RNA molecules Direct amplification of RNA molecules with the enzyme combination of the invention.
  • Tth polymerase 5 U/ ⁇ l in a buffer containing 10 mM po- tas ⁇ ium pH 7.0, 100 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 50% glycerol.
  • a RT reaction mixture (50 ⁇ l) containing 67 mM Tri ⁇ -HCl, pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 50 pmol primer 291 (Amplitest, Cetus Co.), 10 ⁇ g rRNA template, 5 units of Tth polymera ⁇ se and 0,2 units of pyrophosphatase were overlaid with 50 ⁇ l of mineral oil and incubated for 60 min at 65 °C.
  • the thermal profile involved 40 cycle ⁇ of denaturation at 94 °C for 1 min, primer annealing at 56 'c for 1 min, and extension at 72 °C for 2.5 min (for the first cycle for 7 min, and for the last cycle for 10 min) .
  • the re ⁇ ult ⁇ have shown that in the presence of py- rophosphata ⁇ e Tth polymera ⁇ e produced twice as much DNA material using 16S rRNA from Bacillus subtilis as a temp ⁇ late.
  • the enzyme combination of the invention was used for de ⁇ tecting various microorgani ⁇ ms in comparison te ⁇ t ⁇ with Tag-polymerase ("Cetus") .
  • the enzyme combination contained 2.5 unit ⁇ of Tth polymera ⁇ e and 0.125 units of Tth py ⁇ rophosphata ⁇ e.
  • Each test wa ⁇ performed parallel u ⁇ ing Taq- poly era ⁇ e and the enzyme combination according to the invention. Yield of the PCR product in te ⁇ ts with Tag- polymerase wa ⁇ expressed in per cent as a relative quan ⁇ tity, taking the yield obtained using the enzyme combina ⁇ tion according to the invention as 100 %. The number of experiments made is shown in brackets.
  • Herpes simplex type 1 100+7 (4) 49 ⁇ 6 (4)
  • Herpes simplex type 2 100+6 (4) 50+6 (4)
  • the cell ⁇ are grown u ⁇ ing any suitable technique. Briefly, the cells are grown on a medium, in one liter, of nitrilotriacetic acid (100 mg) , tryptone (3 g) , yea ⁇ t extract (3 g) , ⁇ uccinic acid (5 g) , ⁇ odium ⁇ ulfite (50 mg) , riboflavin (1 mg) , K 2 HP0 4 (522 mg) , MgS0 4 (480 mg) , CaCl 2 (222 mg) , NaCl (20 mg) , and trace ele ent ⁇ . The pH of the medium i ⁇ adjusted to 8.0 with KOH.
  • the yield is increased up to 20 g of cells/liter if cultivated with vigorous aeration at a temperature of 70°C.
  • Cells in the late logarithmic growth stage (determined by ab ⁇ orbance at 550 nm) are collected by centrifugation, washed with a buffer and stored frozen at -20°C.
  • the cell ⁇ are thawed, ⁇ u ⁇ pended in a ⁇ uitable buffer ⁇ uch a ⁇ buffer A (10 mM K-pho ⁇ phate buffer, pH 7.4; 1.0 mM EDTA, 1.0 mM beta-vercaptoethanol) , ⁇ onicated and centrifuged.
  • the supernatant is then passed through a column which has a high affinity for proteins that bind to nucleic acid ⁇ , such as Affigel blue column (Biorad) .
  • the enzyme is eluted with a linear gradient, ⁇ uch a ⁇ 0.1 to 2.0 M NaCl buffer A.
  • the peak DNA polymera ⁇ e activity i ⁇ dialyzed and applied to a pho ⁇ phocellulo ⁇ e column.
  • the column i ⁇ wa ⁇ hed and the enzyme activity eluted with a linear gradient ⁇ uch a ⁇ 0.1 to 1.0 M NaCl in buffer A.
  • the fraction ⁇ containing DNA polymera ⁇ e activity are pooled, dialyzed again ⁇ t buffer A, and applied to a high performance liquid chromatography column (HPLC) mono-Q column (anion exchanger) .
  • HPLC high performance liquid chromatography column
  • the fraction ⁇ having thermo ⁇ table polymera ⁇ e activity are pooled, diluted and applied to a HPLC mono-S column (cation exchanger) .
  • Polymerase activity is preferably mea ⁇ ured by the incor ⁇ poration of radioactively labeled deoxynucleotide ⁇ into DNAa ⁇ e-treated, or activated, DNA; following ⁇ ub ⁇ equent ⁇ eparation of the unincorporated deoxynucleotides from the DNA ⁇ ubstrate, polymerase activity is proportional to the amount of radioactivity in the acid-insoluble fraction compri ⁇ ing the DNA (Lehman, I.R., et al., J.Biol.Chem. (1958) 233-163, the di ⁇ closure of which i ⁇ incorporated herein by reference) .
  • the protein pellet was diluted in buffer A + 0.1 M NaCl, dialyzed in the same buffer and applied to a column (2.5 x 10 cm) with DEAE- cellulo ⁇ e.
  • Protein ⁇ were eluted by linear gradient NaCl (0.1-0.3 M NaCl) in buffer A.
  • the fraction ⁇ containing DNA-poly era- se were combined, applied to a column (1.6 x 10 cm) with Blue-Sepharose, and DNA-polymera ⁇ e wa ⁇ eluted with linear gradient NaCl (0.1-1 M NaCl) in buffer A.
  • Fraction ⁇ con ⁇ taining DNA-polymera ⁇ e activity were combined and applied to a column (1.6 x 5 cm) with hydroxy apatite.
  • Protein ⁇ were eluted with linear gradient K-phosphate (0.01-0.5 M) and fraction ⁇ containing DNA-polymera ⁇ e were combined and dialysed in buffer A, contained 0.2 M NaCl, 50% glycerol, 0.1 % Tritone X-100.
  • Enzyme wa ⁇ te ⁇ ted for specific activity diluted to 5 units/ ⁇ l and stored at -20°C.
  • the debris was precipitated by centrifugation at 10 000 g, the supernatant was applied to column of DEAE-sepharose 48 and equilibrated with buffer A.
  • the column wa ⁇ wa ⁇ hed with the same buffer, the proteins were eluted with 0.025 to 0.25 M NaCl gradient (the re ⁇ t of the components a ⁇ in the buffer A) .
  • IP inorganic pyropho ⁇ phata ⁇ e
  • fractions con ⁇ tained inorganic pyrophosphatase were combined and dialyzed against a buffer containing K-phosphate - 10 mM pH-7.4, EDTA - 0.1 mM, DTT - 2 mM, NaCl - 0.2 M, 50% glycerol, 0.1% - ⁇ 100.
  • the reaction mixture (total volume 50 microliter ⁇ ) contain- ing 67 mM Tris-HCL, pH 8.8, 16.6 mM ammonium ⁇ ulphate, 1.5 mM magne ⁇ ium chloride, 0.01% (v/v) Tween-20, 0.25 mM of each dNTP, 1 mg of lambda DNA, 15 pmole ⁇ of each primer, indicated amount ⁇ of enzyme ⁇ , was amplified for 25 cycles at temperature cycling condition ⁇ : 95 °C- 1 min (fir ⁇ t cycle - 2 min) , 56 °C - 1 min (fir ⁇ t cycle - 2 min) , 72 °C - 2.5 min (first cycle - 10 min, second cycle - 5 min, last cycle - 7 min) .
  • the comparative result ⁇ of te ⁇ ting of different enzyme combinations in respect to their ability to amplify DNA- phage ⁇ show that DNA polymerases in any combination in the presence of pyrophosphata ⁇ e are considerably more effective.
  • thermostable pyrophosphatase from Thermus thermophilus to enchance efficiency of coupled reverse transcription/polymera ⁇ e chain reaction (RT/PCR) performed by DNA polymerase from Thermus thermophilus in the pre ⁇ ence of CD-4 mRNA a ⁇ template.
  • RT/PCR coupled reverse transcription/polymera ⁇ e chain reaction
  • RNA ha ⁇ been extracted from CD4 + U937 cell line by u ⁇ ing guanidinium-phenol-chloroform (GPC) method of Chomczyn ⁇ ki and Sacchi (Analytical Biochemi ⁇ try 162, 156- 159 (1987) .
  • GPC guanidinium-phenol-chloroform
  • Cell ⁇ (5 x 10 6 ) were rin ⁇ ed 3 times with cold phosphate-buffered ⁇ aline (PBS) by centrifugation at 1000 x g for 5 min at 4 °C.
  • PBS cold phosphate-buffered ⁇ aline
  • RNA concentration wa ⁇ estimated by UV absorbance at 260 nm.
  • ARl- ⁇ ense primer position on mRNA CD-4 ;145-174) : 5'CAGGGAAACAAAGTGGTGCTGGGCAAA3 ' AR2-anti- ⁇ en ⁇ e primer (po ⁇ ition on mRNA CD-4; 472- 501) : 5'GGTCAGGGTCAGGCTCTGCCCCTGAAGCAG3 ' .
  • a coupled RT/PCR reaction was run a ⁇ de ⁇ cribed by Myers and Gelfand (Biochemistry 30, 7661-7666 (1991) .
  • RT reaction total volume 20 ⁇ l containing 67 mM Tris-HCl pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 1 mM MnCl 2 , 200 ⁇ M each of dATP, dCTP, dGTP, dTTP, 15 pmol primer anti-sen ⁇ e (AR2) , 200 ng of total cellular RNA as template, 5 units of DNA polymerase from Thermus thermophilus and indicated amounts of pyropho- ⁇ phatase ⁇ from Thermus thermophilus or Thermus aquaticus .
  • RT reaction mixture ⁇ were overlaid with 70 ⁇ l of mineral oil and incubated at 70 °C for 15 min. After incubation the ⁇ amples were placed on ice.
  • PCR mixture (total volume 80 ⁇ l) containing 67 mM Tri ⁇ - HC1, pH 8.8, 16.6 mM (NH 4 ) 2 S0 4 , 0.75 mM EDTA, 0.025 % (v/v)
  • Figure 6 shows the yield of specific 356-bp product after RT/PCR using the following enzyme combinations:

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Abstract

La présente invention concerne une composition d'enzymes thermophiles en tant que moyen pour la synthèse in vitro d'ADN. La composition comprend, en combinaison, une enzyme pouvant effectuer la polymérisation du dNTP sous forme de molécules d'ADN synthétisées de nova dans un intervalle de température de 56 à 90 °C (ADN-polymérase thermophile) et une enzyme pouvant éliminer le pyrophosphate (pyrophosphatase thermophile) dans le même intervalle de température. L'invention concerne également la préparation et l'utilisation de ladite composition.
PCT/FI1993/000340 1992-09-01 1993-08-31 Synthese in vitro de molecules d'adn Ceased WO1994005797A1 (fr)

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AU49608/93A AU4960893A (en) 1992-09-01 1993-08-31 Synthesis of dna molecules in vitro

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FI923911A FI923911A7 (fi) 1992-09-01 1992-09-01 DNA-molekyylien in vitro-synteesi

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0745676A1 (fr) * 1995-05-31 1996-12-04 Amersham Life Science Inc Polymérase d'ADN thermostable
US5665551A (en) * 1995-09-13 1997-09-09 Roche Molecular Systems, Inc. Purified nucleic acid encoding a thermostable pyrophosphatase
WO1997037038A1 (fr) * 1996-03-29 1997-10-09 Boehringer Mannheim Gmbh Procede de multiplication specifique d'acides amines longs par pcr
WO1998015655A1 (fr) * 1996-10-07 1998-04-16 The Perkin-Elmer Corporation Reaction d'extension d'amorce utilisant une paire d'enzymes d'un meme substrat pour consommer le pyrophosphate
US6001645A (en) * 1995-06-07 1999-12-14 Promega Corporation Thermophilic DNA polymerases from thermotoga neapolitana
US6077664A (en) * 1995-06-07 2000-06-20 Promega Corporation Thermophilic DNA polymerases from Thermotoga neapolitana
US6107032A (en) * 1996-12-20 2000-08-22 Roche Diagnostics Gmbh Method for the direct, exponential amplification and sequencing of DNA molecules and its application
US6238905B1 (en) 1997-09-12 2001-05-29 University Technology Corporation Thermophilic polymerase III holoenzyme
US6291164B1 (en) 1996-11-22 2001-09-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
US6406891B1 (en) 1998-09-28 2002-06-18 Board Of Regents, The University Of Texas System Dual RT procedure for cDNA synthesis
GB2377991A (en) * 2001-04-30 2003-01-29 Secr Defence Brit DNA amplification in the presence of a pyrophosphate salt and a pyrophosphatase
US6605428B2 (en) 1996-12-20 2003-08-12 Roche Diagnostics Gmbh Method for the direct, exponential amplification and sequencing of DNA molecules and its application
US6677146B1 (en) 2000-03-28 2004-01-13 Replidyne, Inc. Thermophilic polymerase III holoenzyme
WO2005033328A3 (fr) * 2003-09-30 2006-04-06 Perkinelmer Las Inc Compositions et procedes de genotypage de polymorphismes d'un nucleotide simple
US8927211B2 (en) 2005-02-09 2015-01-06 Pacific Biosciences Of California, Inc. Nucleotide compositions and uses thereof
EP2876166A1 (fr) 2013-11-20 2015-05-27 Roche Diagniostics GmbH Nouveaux composés pour le séquençage par synthèse
GB202007428D0 (en) 2020-05-19 2020-07-01 Fabricnano Ltd Polynucleotide synthesis
GB202114105D0 (en) 2021-10-01 2021-11-17 Fabricnano Ltd Nucleotide synthesis
CN114085891A (zh) * 2021-11-23 2022-02-25 广州达安基因股份有限公司 基于重组酶聚合酶扩增技术的逆转录扩增系统和方法

Citations (1)

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WO1990012111A1 (fr) * 1989-04-12 1990-10-18 President And Fellows Of Harvard College Reactions d'extension d'amorce ameliorees

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DIALOG INFORMATION SERVICES, File 154, MEDLINE, Dialog Accession No. 07020198, HOHNE W.E. et al., "Kinetic Characterization of a Thermostable Inorganic Pyrophosphatase from Thermus Thermophilus"; & BIOMED BIOCHIM ACTA, 1988, 47 (12), p. 941-7. *
NUCLEIC ACIDS RESEARCH, Volume 17, No. 24, 1989, T.A. BECHTEREVA et al., "DNA Sequencing with Thermostable Tet DNA Polymerase from Thermus Thermophilus", page 10507. *

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996038568A1 (fr) * 1995-05-31 1996-12-05 Amersham Life Science, Inc. Adn-polymerases thermostables
EP0745676A1 (fr) * 1995-05-31 1996-12-04 Amersham Life Science Inc Polymérase d'ADN thermostable
US5885813A (en) * 1995-05-31 1999-03-23 Amersham Life Science, Inc. Thermostable DNA polymerases
EP0873420A4 (fr) * 1995-06-07 2003-09-03 Promega Corp Adn polymerases de nature thermophile, issues de thermotoga neapolitana
US6001645A (en) * 1995-06-07 1999-12-14 Promega Corporation Thermophilic DNA polymerases from thermotoga neapolitana
US6077664A (en) * 1995-06-07 2000-06-20 Promega Corporation Thermophilic DNA polymerases from Thermotoga neapolitana
US5665551A (en) * 1995-09-13 1997-09-09 Roche Molecular Systems, Inc. Purified nucleic acid encoding a thermostable pyrophosphatase
EP0763599A3 (fr) * 1995-09-13 1999-09-01 F. Hoffmann-La Roche Ag ADN codant une pyrophosphatase thermostable
WO1997037038A1 (fr) * 1996-03-29 1997-10-09 Boehringer Mannheim Gmbh Procede de multiplication specifique d'acides amines longs par pcr
WO1998015655A1 (fr) * 1996-10-07 1998-04-16 The Perkin-Elmer Corporation Reaction d'extension d'amorce utilisant une paire d'enzymes d'un meme substrat pour consommer le pyrophosphate
US6291164B1 (en) 1996-11-22 2001-09-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
US7344835B2 (en) 1996-11-22 2008-03-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
US6764839B2 (en) 1996-11-22 2004-07-20 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
US6605428B2 (en) 1996-12-20 2003-08-12 Roche Diagnostics Gmbh Method for the direct, exponential amplification and sequencing of DNA molecules and its application
US6107032A (en) * 1996-12-20 2000-08-22 Roche Diagnostics Gmbh Method for the direct, exponential amplification and sequencing of DNA molecules and its application
US6238905B1 (en) 1997-09-12 2001-05-29 University Technology Corporation Thermophilic polymerase III holoenzyme
US6406891B1 (en) 1998-09-28 2002-06-18 Board Of Regents, The University Of Texas System Dual RT procedure for cDNA synthesis
US6677146B1 (en) 2000-03-28 2004-01-13 Replidyne, Inc. Thermophilic polymerase III holoenzyme
WO2002088387A3 (fr) * 2001-04-30 2003-12-11 Secr Defence Procede d'amplification
KR101039563B1 (ko) * 2001-04-30 2011-06-09 더 세크러터리 오브 스테이트 포 디펜스 증폭 방법
US6951744B2 (en) 2001-04-30 2005-10-04 The Secretary Of State For Defence Amplification process
GB2377991B (en) * 2001-04-30 2004-01-28 Secr Defence Brit DNA amplification in the presence of a pyrophosphate salt and a pyrophosphatase
CN1318604C (zh) * 2001-04-30 2007-05-30 英国国防部 扩增方法
GB2377991A (en) * 2001-04-30 2003-01-29 Secr Defence Brit DNA amplification in the presence of a pyrophosphate salt and a pyrophosphatase
US7449312B2 (en) 2001-04-30 2008-11-11 The Secretary Of State For Defense In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Kit for conducting a polymerase chain reaction
WO2005033328A3 (fr) * 2003-09-30 2006-04-06 Perkinelmer Las Inc Compositions et procedes de genotypage de polymorphismes d'un nucleotide simple
US8927211B2 (en) 2005-02-09 2015-01-06 Pacific Biosciences Of California, Inc. Nucleotide compositions and uses thereof
EP2876166A1 (fr) 2013-11-20 2015-05-27 Roche Diagniostics GmbH Nouveaux composés pour le séquençage par synthèse
GB202007428D0 (en) 2020-05-19 2020-07-01 Fabricnano Ltd Polynucleotide synthesis
WO2021234378A1 (fr) 2020-05-19 2021-11-25 FabricNano Limited Synthèse de polynucléotides
GB202114105D0 (en) 2021-10-01 2021-11-17 Fabricnano Ltd Nucleotide synthesis
CN114085891A (zh) * 2021-11-23 2022-02-25 广州达安基因股份有限公司 基于重组酶聚合酶扩增技术的逆转录扩增系统和方法

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AU4960893A (en) 1994-03-29
FI923911A0 (fi) 1992-09-01
FI923911A7 (fi) 1994-03-02

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