SYNTHESIS OF DNA MOLECULES IN VITRO
ABSTRACT
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.
BACKGROUND OF THE INVENTION
After the properties of DNA-polymerase I from Esche- richia coli were first reported by A. Kornberg (Watson et al., "Recombinant DNA", Cold Spring Harbor Laboratory, 1983) , many laboratories started investigations on the mechanism of the DNA synthesis. Reproduction of this pro¬ cess jLn vitro opened great possibilities for basic re¬ search and provided solutions for many applied problems.
A more powerful method of enzymatic synthesis of DNA mole- cules in vitro has been described by Saiki et al. (Scie¬ nce, 1988, 239, 487-491). This method (given the name of polymerase chain reaction - PCR) allows amplification of any desired specific nucleotide sequences contained in a nucleic acid or a mixture thereof. The process involves hybridizing oligonucleotide primers to separate comple¬ mentary strands of nucleic acids and extending the primers to form complementary primer extension products which then act as templates for synthesizing the desired nucleic acid sequence. Individual stages of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desirable. The above methodology has some limitations. For example, during analysis of a complex mixture of nucleic acids of various origin and containing a desirable sequence of DNA, it is impossible to amplify target DNA during the theoreti- cally calculated number of cycles. In these cases, it is extremely important to increase the efficiency of PCR, especially in the application of PCR for diagnostic purposes, in particular, in the diagnosis of latent virus infections, when it is necessary to identify a single copy of viruses, or in analysis of a mixture of microorganisms from the environment to detect an etiologic agent of infectious diseases.
Methods enabling direct analysis of RNA molecules are of great importance for detecting oncogene expression. In clinical practice the 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.
Also the 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. The primer iε elongated using a DNA polymerase and each reaction terminated at a specific base (guanine, G, adenine, A, thymine, T, or cytosine, C) via the incorporation of an appropriate chain terminating agent, 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.
There are two main problems in using the DNA-sequencing method:
I. 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.
II. 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
Kornberg, A., J. Biol. Chem. 1969, 244 , 3019-3028) . Tabor S. and Richardson C. have shown (J. Biol. Chem., 1990, 265, 8322-8328) that when a DNA sequencing reaction is carried out with 28T7 DNA polymerase, specific bands disappear with time of incubation, despite the absence of exonuclease activity. They showed that this is the result of pyrophos¬ phorolysis by PPi, a product of DNA synthesis, which attacks the 3'-terminal internucleotide phosphate in a reversal of the polymerase reaction, releasing ddNTP and degrading the DNA chain by one nucleotide. Once a ddNMP has been removed, the primer iε extended past the site due to the high ratio of dNTPs to ddNTPε, and the original band disappears.
The above information shows that molecular biology methods based on DNA syntheεis in vitro are widely used in funda- mental and applied research and any improvementε in these methods would be of great importance.
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 analyεis of the many publications dealing with the application of PCR for diagnostic purposeε 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 oεtability of recombinant DNA- polymerases by protein engineering methodε or to screening new thermoresistant enzymes.
SUMMARY OF THE INVENTION
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 εensi- tivity is that during DNA-synthesis some productε 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 poεεibility 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.
Thus the present invention aims at removing εuch accumu¬ lated products, in particular pyrophosphate formed, by using a combination of enzymes, εpecifically a thermophi¬ lic DNA polymeraεe enzyme in combination with a thermo¬ philic pyrophoεphataεe enzyme.
Specifically the invention concernε the combination of two thermophilic enzymeε and uεe thereof aε a tool for nucleic acid εyntheεiε 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 eraεe, and the other being able to eliminate accumulated py- rophoεphate within the εame temperature interval, i.e. a thermophilic pyrophoεphatase.
The invention also concerns an improvement in a method for in vitro enzymatic synthesiε of nucleic acid molecules, the improvement consiεting in that a thermophilic DNA-polyme¬ raεe iε used which is able to carry out the polymerization of dNTP within the temperature interval of 56-90 °C, in combination with a thermophilic pyrophosphataεe which is able to eliminate accumulated pyrophoεphate within the same temperature interval.
Thus the invention makes it poεsible to effectively crea- te n vitro de nova syntheεized DNA. DETAILED DESCRIPTION OF THE INVENTION
We have εtudied the inhibitory effect of exogenic py- rophoεphate on amplification of the lambda DNA fragment. We have then demonεtrated that the concentration of py¬ rophosphate during the PCR varies from 0.2 to 0.5 mM, when the number of cycles iε from 20 to 30. Experimental inveεtigationε further demonεtrated that in thiε interval of concentrationε εignificant or complete inhibition of the reaction waε obεerved, aε iε εhown in the appended Fig. 2.
According to the invention we have shown that the inhibi¬ tory effect of pyrophosphate is very easy to avoid by the addition of pyrophosphataεe from E . coli . Thiε fact evi- denceε the inhibitory role of pyrophosphate in PCR. This enzyme is, however, not thermoεtable enough, wherefore we iεolated thermophilic pyrophoεphataεe i.a. from Thermus thermophilus (Tth pyrophosphatase) and used it in combina¬ tion with polymeraεe from Thermus thermophilus (Tth polymerase) for amplification. Experiments demonstrated that such a combination of thermophilic enzymes allows for effective amplification at a pyrophoεphate concentration of 0.5 mM.
Pyrophoεphate concentrationε of 0.2 to 0.5 mM can modula¬ te the DNA-polymeraεe function and lead to premature ter¬ mination of the DNA synthesiε. Thiε can prevent effective amplification of a long DNA fragment. Experimental data confirm our findingε and demonstrate that amplification of a lambda DNA fragment 10 kilobaεeε long iε carried out more effectively in caεe the enzyme combination of the invention waε uεed. We have according to the invention been able to incorporate twice the amount of labelled dNTP into the εyntheεized DNA in the presence of pyrophosphatase.
The modulating effect of a high concentration of py¬ rophosphate on the functions of DNA polymeraεe can alεo lead to decreasing fidelity and be accompanied with an increaεe of error numberε during the DNA εynthesis. Ac¬ cording to the invention, the application of pyrophospha¬ tase in combination with DNA-polymerase pro oteε high fidelity of DNA synthesiε.
Thuε, the above mentioned data convincingly demonstrate that the application of pyrophoεphataεe can εignificantly improve DNA synthesiε in vitro. The enzyme combination according to the invention can advantageously be used alεo for DNA sequence analysiε to read a εtrong εecondary εtructure region and aε a solution of the problems related to the accumulation of pyrophosphate.
Aε Tth polymeraεe haε reverεe tranεcriptase activity, the possibility of application of the enzyme combination ac¬ cording to the invention for direct amplification of RNA moleculeε haε alεo been inveεtigated. Such approach gives an opportunity of omitting the step of copy DNA syntheεiε with the help of AMV (Avian Myeloblaεtoεiε Virus, a com¬ mercial product of Pharmacia) reverse transcriptaεe. Many of the problemε aεεociated with a εtrong εecondary εtruc¬ ture of RNA are minimized by uεing a thermoεtable DNA polymerase for reverse tranεcription, in which caεe pre- dominantly full-length products can be obtained. cDNA can be amplified in the polymerase chain reaction with the same enzyme.
For thiε purpoεe we have εtudied the uεe of Tth polymera- εe in combination with pyrophoεphataεe in reverse trans¬ cription reaction and PCR amplification (RT/PCR) . The application of the RT/PCR technique may be successful in various areas, in particular, in the εtudy of viruε mole¬ cular εtructures as well as in the detection, quantifica- tion and cloning of cellular and viral RNA. The enzyme mixture has been used for RT/PCR with three different templates: pAWIOS RNA template from Gene AmpR PCR kit ("Cetuε") , 16S RNA from Bacillus εubtilis and Influenza viruε type A RNA. The experimental data indicated suc¬ cessful application of the enzyme combination of the in¬ vention in RT/PCR.
All of the foregoing problems are εolved in the preεent invention, aε defined above.
According to the invention it is possible to overcome the present restrictionε on PCR, and for example to increaεe the number of cycleε, εuch aε 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 iε alεo possible to determine succesεively different agentε in one and the same sample, as no pyrophosphate is accumulated in the sample. Thiε iε of courεe an advantage e.g. when there is a limited availability of sample material.
According to the invention it iε alεo poεsible to provide, due to the permanent intensity of bands, low background level and high precision, sequencing syεtems 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 aε amplified DNA, big double-εtranded DNA-templateε (such aε lambda) , GC-rich templates and long poly(A) tails.
The first component in the combination according to the invention iε a thermophilic DNA-polymeraεe from a ther¬ mophilic microorganiεm, εuch aε Thermus thermophilus mic- roorganiεms. Also other thermophilic microorganisms may be used as the enzyme εource, such aε Thermus aquaticus and Thermus ruber.
The genus Thermus is deεcribed in Buchanan and Gibbons (editors) Bergey'ε Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore, p.295. See also T . thermophilus HB8 , Sato S., et al., (1978) J.Bio¬ chem. 84: p.1319; T . aquaticus YT1 , Sato S., et al., (1977), Proc. Natl. Acad. Sci. USA, V. 74, p. 542-546. Thermus ruber haε been published by Loginova in "Thermo¬ philic strainε of microorganiεmε" (Ruεεian) .
Commercially available 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 Viruε uεing DNA-polymerase from Thermus thermophilus . 1990, Mol. Cell. Probes, Vol. 4, pp.435-443; Barballeira, N. et al., Purification of a Thermostable DNA-polymerase from Thermus thermophilus HB- 8 Uεeful in the Polymeraεe Chain Reaction. 1990, Biotechni- ques, V. 9, pp. 276-281); Lawyer F. C. et al., Isolation, Characterization and Expreεεion in Escherichia coli of the
DNA Polymeraεe Gene from Thermus aquaticus , J. Biol. Chem., (1989) 264, No. 11 , 6427; Engelke, D. R et al., Purification of Thermus aquaticus DNA Polymeraεe Expreεεed in Escherichia coli , Anal. Biochem. (1990) 191, 396; Chien, A. et al., Deoxyribonucleic Acid Polymeraεe from the Extremely Thermophilic Thermus aquaticus . 1987, J. Bacte- riol. Vol. 127, pp 1550-1557; Kaledin, A.S. et al., Iεolation and Properties of DNA Polymerase from the Extremely Thermophilic Bacterium Thermus aquaticus YT 1, 1980, Biokhimya, Vol. 45, pp. 644-651 (Ruεεian) ; Kaledin, A.S. et al., Iεolation and Propertieε of DNA-polymeraεe from the Extremely Thermophilic Bacterium Thermus ruber . 1981, Biokhimya, Vol. 47, pp. 1785-1791 (Russian) ) .
A main property of these enzymes is that they do not loo¬ se their activity at high temperature and can be uεed to solve secondary structure problems in the DNA sequencing analysis and in PCR. As will be shown later, the enzyme source is not critical as long as the enzymes obtained exhibit the required properties. In addition, the polyme¬ rase enzyme has the distinctive feature that it iε able to make copy DNA uεing an RNA template becauεe it possesses reverse transcriptase activity.
The εecond component of the enzyme combination iε a ther¬ mophilic pyrophoεphataεe, i.e. inorganic pyrophoεphataεe (IP) which iε able to eliminate accumulated pyrophosphate by pyrophosphorolyεis. As shown above, the accumulation of pyrophosphate during the DNA-syntheεis reaction inhibitε DNA-polymeraεe activity and decreaεeε the efficiency of the reaction.
The thermophilic pyrophoεphataεe for the purpoεes of thiε invention iε preferably isolated from Thermus thermophi¬ lus , but can also be obtained from other thermophilic icroorganismε, εuch aε Thermus aquaticus and Thermus rujbe .
According to a preferred embodiment, the thermophilic polymerase and the thermophilic pyrophosphataεe are both iεolated during the same procedure from the same mic- roorgansim, such aε Thermus thermophilus . In εuch a pro¬ cedure the bacterial cellε are diεintegrated e.g. by ult- raεonication in a suitable buffer (e.g. buffer A) , and the supernatant obtained after centrifugation iε chroma- tographed on a suitable ion exchange column, such as DEAE- Sepharose, in order to separately elute the pyrophosphata¬ se and the DNA polymerase, using a suitable eluent gra¬ dient, such as a gradient of 0.025-0.25 M NaCl and testing for reεpective enzyme activity in the eluate. The two enzymes may be eluted with εuitable NaCl gradients, such as those discloεed in the Examples. The pyrophosphatase and the DNA-polymeraεe fractions are pooled reεpectively, and thereafter purified individually, εuch as by ion exhange chromatography or by hydrophobic chromatography.
In the following the isolation and purification of DNA- polymerase and pyrophosphataεe from Thermus thermophilus iε illustrated by means of a flow diagram.
1. Ultrasonic deεtruction of bacterial cellε
2 . Chromatography on IDEAE-Sepharoεe 4B
Pyrophoεp /hatase DNA-polymeraεe
Chromato Igraphy on Chrom Iatography on DEAE-Toyopearl 65F (Toyo) phoεphocelluloεe P-ll (Whatman)
Chromato Igraphy on Chrom iatography on butyl-Toyopearl 65F (Toyo) hydroxylapatite (BioRad)
Chrom Iatography on Toyopearl HW-60F (Toyo)
Detailε of the iεolation and purification εteps are given in the exampleε.
In carrying out the PCR reaction according to the inven¬ tion, per one unit (U) of polymeraεe activity, a wide range of pyrophoεphatase activities can be used, such as e.g. from 0.04 to 0.5 U. A εuitable 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 μl, iε 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 μl .
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 conditionε: 25 mM TAPS pH 9.3 (at 25 °C) ; 50 mM KCl, 2 mM MgCl2, 1 mM jS-mer- captoethanol; 200 μM each of dATP, dGTP, dTTP; 100 μM dCTP (a mixture of unlabelled and α-[32P]-labelled) ; 12.5 μg of activated DNA of εalmon εperm, final volume 50 μl.
The pyrophosphatase activity unit is the enzyme amount that produces 2 μmol of phoεphate from pyrophoεphate in 1 min at +75 °C under the following conditionε: 1 mM pyrophosphate, 2 mM MgCl2, 50 mM Triε HC1 pH 9.0 (at + 25°C) .
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawing,
Fig.l illuεtrateε a compariεon of thermoεtability of pyrophoεphataεe from Thermus thermophilus and Escherichia coli with reεpect to the number of cycleε in PCR. Fig. 2 illuεtrates amplification of lambda DNA in the presence of different concentrations of pyrophosphataεe under uεual PCR conditionε for amplification of 500 bp fragment of lambda DNA.
Fig. 3 illuεtrateε the εyntheεiε of a 10 kilobase fragment of lambda DNA and the effect of using the enzyme combina- tion.
Fig. 4 illustrateε incorporation of P dNTP into synt¬ hesized DNA in PCR with and without pyrophosphataεe from Thermus thermophilus . Fig. 5 illustratesthe efficiency of amplification of a 8 kb lambda DNA sequence uεing different enzyme combinations.
Fig. 6 illustrateε the efficiency of uεing RT/PCR using CD 4 mRNA as template.
Experimental data illuεtrating the principal pointε of the invention, are preεented below. Example 1 .
Iεolation and εeparation of pyrophoεphatase and DNA-poly¬ merase from Thermus thermophilus
1. Isolation of the enzymes
50 g of biomaεε of Thermus thermophilus were εuspended in 100 ml of buffer A (0.02 M Tris.HCl pH 7.8, 25 m NaCl, 0.001 M EDTA, 2 mM DTT) and when kept on ice, the εuεpen- εion waε diεintegrated by ultraεonication for 30 min.
The debriε was precipitated by centrifugation at 10 000 g, the supernatant was applied to a column (2.6 x 40 cm) of DEAE-Sepharoεe 4B, equilibrated with buffer A. The column waε waεhed with the εame buffer (about 600 ml) , the proteinε were eluted with 0.025 to 0.25 M NaCl gradient (the reεt of the componentε aε in the buffer A) .
In the eluate, the activity of Tth polymeraεe and inor- ganic pyrophoεphataεe (IP) were assayed. Tth polymerase was eluted with 0.09-0.15 M NaCl, and IP was eluted with 0.13- 0.16 M NaCl.
The IP-containing fractions were pooled, and the purifi- cation procedure was thereafter performed individually for Tth polymerase and IP.
2. IP-purification
The 0.13-0.16 M NaCl fractions received from the DEAE- Sepharoεe 4B column were pooled, dialyzed againεt 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- tionε were collected and uεed for further purification. The IP-containing fractionε were combined and salted out with (NH4)2S04, the latter being added up to 75 % saturation. The sediment was disεolved in 1.5 M (NH4)2SO. (the rest of the componentε except (NH4)2S0. were as in buffer A) and applied to a column (1.6x20 cm) of butyl-Toyopearl 65F. The proteinε were eluted with a 1.5 -0.25 M (NH4)2S04 gradient. The IP-containing fractionε were combined, concentrated by deεalting with (NH4)2S04 to 75%, diεεolved in a buffer containing 1.5 M (NH4)2S04, 0.1 mM EDTA, 2 mM DTT and εtored at +4°C.
3. Purification of Tth polymeraεe Fractionε obtained from the DEAE-Sepharoεe 4B and DEAE- Toyopearl 65F were combined, dialyzed againεt a buffer containing 0.02 M K-phoεphate 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 fractionε (0.3-0.4 M NaCl) were combined and applied to a column (1.6x20 cm) of hydroxya- patite HT (BioRad, Biogel HT) , the proteinε were eluted with a 0.02-0.25 M K-phoεphate gradient, pH 7.0.
The polymerase-containing fractionε were combined and chromatographed on a 2.6x60 cm column of Toyopearl HW-60F. The active fractionε were combined, concentrated and dialyzed against a buffer containing 0.1 M NaCl, 0.02 M K- phoεphate, pH 7.0, 0.1 mM EDTA, 2mM DTT, 50 % (w/w) glycerol, and εtored at -20°C.
The respective yield from 50 g of biomaεs was as follows: Tth polymeraεe 25000 units, inorganic pyrophosphataεe 3000 units. Example 2 .
Compariεon of the thermoεtability of pyrophoεphataεe from Thermus thermophilus and Escherichia coli in the enzyme mixture.
Enzyme activity waε assayed in a medium containing 50 mM Tris.HCl pH 9.0, 2 mM MgCl2, 1 mM pyrophosphate. The unit of activity is the amount of enzyme needed for the tranε- formation of 1 mM pyrophoεphate to phoεphate during 1 minute. The method of pyrophosphate aεsay is based on a colour reaction, which is run after the interaction of phosphate with Na olybdate. In the reaction phosphomolyb- date iε formed followed by reduction with εtannic chloride.
Two enzyme εamples were analysed:
1. DNA polymeraεe pluε pyrophoεphataεe both from Thermus thermophilus
2 . DNA polymeraεe from Thermus thermophilus pluε py- rophosphatase from Escherichia coli .
The enzymes were incubated in PCR buffer (67 irύ Tris.HCl, pH 8.8; 16 mM (NH4)2S04; 1.5 mM MgCl2; 0.01 % Tween-20) .
The PCR cycle consisted of three stages:
1. +94 'C - 1 min
2. +56 °C - 1 min
3. +72 'C - 2,5 min
The reεults are shown in the appended Fig. 1. The Figure shows that Tth pyrophosphataεe retained about 70% of its activity after 20 cycleε, whereas at the same time E . coli pyrophoεphataεe loεt itε activity completely. Example 3 .
Accumulation of pyrophoεphate during PCR.
In the proceεε of PCR, utilization of dNTP leads to the accumulation of pyrophosphate. Calculations εhow that the concentration of pyrophoεphate reacheε the critical point (0.5 mM) which can inhibit DNA polymeraεe after 20 cycles.
For confirmation of these data the following experiments were performed. A fragment of lambda DNA was amplified by Tth polymerase in the presence of various concentrationε of pyrophoεphate (the conditionε of PCR aε in the Example 1) . The results, which are indicated in the Fig. 2, illu- strate the entire inhibition of the amplification proceεs at a pyrophosphate concentration of 0.5 mM. This inhibi¬ tory effect was not observed with the invented enzyme mixture (sampleε 4, 6 and 8) .
Example 4.
Syntheεiε of a 10 kilobaεe fragment of lambda DNA. Effect of uεing the enzyme combination of the invention.
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 sampleε contained 0.2 units of Tth pyrophosphatase. Amplification was performed as follows:
94 °C - 1 min
56 °C - 1 min 1 cycle 72 °C - 10 min
94 °C - 0,5 min 56 °C - 0.5 min 18 cycleε 72 °C - 1,5 min 94 °C - 1 min
56 °C - 1 min 1 cycle
72 °C - 7 min
The results as demonstrated in the Fig. 3 εhowed the moεt effective amplification in the pyrophosphatase containing sample (εampleε 2 and 4) .
Example 5.
Incorporation of 32P dNTP into the εynthesized DNA during PCR in the presence of pyrophoεphatase from Thermus ther¬ mophilus .
The conditionε of the experiment are the same aε in the previouε example. The experimental reεults which are de¬ monstrated in the Fig. 4 εhow that after 20 cycles of amplification, incorporation of labelled dNTP was 2.5 timeε higher in the pyrophoεphatase-containing sample.
Example 6
Application of the enzyme mixture of the invention in a procedure for sequencing DNA as follows (method according to Sanger et al. supra):
I. Annealing step 10 μl of mixture containing: 0.1 μl of single-stranded DNA, 1 μl of sequencing buffer,
5 μg of single-stranded DNA primer, 15 to 30 baseε in length
The reaction mixture waε incubated for one minute at 93 "C followed by 20 minutes at 55 °C. II. Labelling εtep
To the reaction mixture was added:
1 μl of 0.75 mM solution of dGTP, dCTP and dTTP, 0.5 μl of 35-S-thio dATP, 1 μl of the enzyme combination of the invention (2 unitε of DNA-polymeraεe and 0.1 unit of pyrophoεphatase) . The mixture waε incubated for five minuteε at 42 °C.
III. Sequencing step 2 μl of the resulting mixture were added to four tubes with
2 μl ddN/dN mixtures and heated for three minutes at 72 'C. The ddN/dN mixture was prepared in 25 M Triε.HCl and 7 mM MgCl2 and contained the following:
G mix - 160 μM ddG, 10 μM each dNTP
A mix - 500 μM ddA, 10 μM each dNTP
T mix - 500 μM ddT, 10 μM each dNTP
C mix - 300 μM ddC, 10 μM each dNTP
IV. Termination εtep
2 μl of a formamide dye mix were added and the mixture was heated three minutes at 90 °C before loading on gel. The sequencing buffer used in step 1 is made up of'25 mM TrisHCl pH 8.8; 1 mM MgCl2, 0.1% Nonidet P40 and 0.1% Twe- en-20.
Application of the enzyme combination made it posεible to avoid problemε with the εecondary εtructure of DNA temp¬ late and allowed the accumulation of pyrophoεphate.
Example 7.
Direct amplification of RNA molecules with the enzyme combination of the invention. RT/PCR aεsay of 16S rRNA from Bacillus subtilis .
Components:
Tth polymerase, 5 U/μl in a buffer containing 10 mM po- tasεium pH 7.0, 100 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 50% glycerol.
Thermophilic pyrophoεphataεe from Thermus thermophilus AMV Reverεe Transcriptase (from "Pharmacia")
165 rRNA from Bacillus subtilis, 10 μg/ml
Primerε:
#1 - 5' AAGGAGGTGATCCAGCCGCACCTCC-3' #2 - 5' AGACTTTGATCCTGGCTCAG-3' dATP, dCTP, dGTP, dTTP, 10 mM εolution 10 x reaction buffer, containing 670 mM Triε.HCl, pH 8.8,
166 mM (NH4)2S04
0.1% Tween-20, 15 mM MgCl2
A RT reaction mixture (50 μl) containing 67 mM Triε-HCl, pH 8.8, 16.6 mM (NH4)2S04, 50 pmol primer 291 (Amplitest, Cetus Co.), 10 μg rRNA template, 5 units of Tth polymera¬ se and 0,2 units of pyrophosphatase were overlaid with 50 μl of mineral oil and incubated for 60 min at 65 °C. Fol¬ lowing the RT reaction, aliquots (10 μl) were added into the PCR reaction mixture (50 μl) , containing 67 mM Tris.¬ HCl, pH 8.8, 16.6 mM (NH4)2S04, 0.01% Tween-20, 1.5 mM MgCl2, 200 μmol each of dATP, dCTP, dGTP, dTTP, 50 pmol primer 1 and 50 pmol primer 2, 2.5 units of Tth polymera¬ se and 0.2 unitε of pyrophoεphataεe and overlaid with 50 μl of mineral oil. The samples were then amplified in a Perkin-Elmer Cetus Instrumentε DNA Thermal Cycler. The thermal profile involved 40 cycleε 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) .
Aliquotε (5 μl) of the PCR amplification were analyzed by electrophoresis in a 0,8% (w/v) agarose gel εtained with ethidium bromide.
The reεultε have shown that in the presence of py- rophosphataεe Tth polymeraεe produced twice as much DNA material using 16S rRNA from Bacillus subtilis as a temp¬ late.
Example 8.
The enzyme combination of the invention was used for de¬ tecting various microorganiεms in comparison teεtε with Tag-polymerase ("Cetus") . The enzyme combination contained 2.5 unitε of Tth polymeraεe and 0.125 units of Tth py¬ rophosphataεe. Each test waε performed parallel uεing Taq- poly eraεe and the enzyme combination according to the invention. Yield of the PCR product in teεts with Tag- polymerase waε 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.
Microorganiεm Yield of PCR product (%) Enzyme combination racr-polvmerase
Chlamydia trachomatiε 100+7 (3) 40+4 (3)
HIV-1 100±9 (5) 34±3 (5)
Epεtein-Barr 100±8 (5) 45+5 (5)
Herpes simplex type 1 100+7 (4) 49±6 (4)
Herpes simplex type 2 100+6 (4) 50+6 (4)
Staphylococcus aureus
Enterotoxin A 100+10 (3) 37+2 (3)
Enterotoxin B 100+9 (3) 42±4 (3) Example 9 .
Purification of DNA-polymeraεe from Thermus aquaticus
For recovering the native protein the cellε are grown uεing any suitable technique. Briefly, the cells are grown on a medium, in one liter, of nitrilotriacetic acid (100 mg) , tryptone (3 g) , yeaεt extract (3 g) , εuccinic acid (5 g) , εodium εulfite (50 mg) , riboflavin (1 mg) , K2HP04 (522 mg) , MgS04 (480 mg) , CaCl2 (222 mg) , NaCl (20 mg) , and trace ele entε. The pH of the medium iε 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 abεorbance at 550 nm) are collected by centrifugation, washed with a buffer and stored frozen at -20°C.
The cellε are thawed, εuεpended in a εuitable buffer εuch aε buffer A (10 mM K-phoεphate buffer, pH 7.4; 1.0 mM EDTA, 1.0 mM beta-vercaptoethanol) , εonicated and centrifuged. The supernatant is then passed through a column which has a high affinity for proteins that bind to nucleic acidε, such as Affigel blue column (Biorad) . The nucleic acids present in the supernatant solution of T . aquaticus and many of the proteins pasε through the column and are thereby removed by waεhing the column with several column volumes of low εalt buffer at pH of about 7.0. After washing, the enzyme is eluted with a linear gradient, εuch aε 0.1 to 2.0 M NaCl buffer A. The peak DNA polymeraεe activity iε dialyzed and applied to a phoεphocelluloεe column. The column iε waεhed and the enzyme activity eluted with a linear gradient εuch aε 0.1 to 1.0 M NaCl in buffer A. The peak DNA polymeraεe activity iε dialyzed and applied to a DNA celluloεe column. The column iε waεhed and DNA polymeraεe activity iε eluted with a linear gradient of 0.1 to 1.0 M NaCl in buffer A. The fractionε containing DNA polymeraεe activity are pooled, dialyzed againεt buffer A, and applied to a high performance liquid chromatography column (HPLC) mono-Q column (anion exchanger) . The enzyme iε again eluted with a linear gradient εuch aε 0.05 to 1.0 M NaCl in a buffer A. The fractionε having thermoεtable polymeraεe activity are pooled, diluted and applied to a HPLC mono-S column (cation exchanger) . The enzyme iε again eluted with a linear gradient, εuch as 0.05 to 1.0 M NaCl in buffer A.
Polymerase activity is preferably meaεured by the incor¬ poration of radioactively labeled deoxynucleotideε into DNAaεe-treated, or activated, DNA; following εubεequent εeparation of the unincorporated deoxynucleotides from the DNA εubstrate, polymerase activity is proportional to the amount of radioactivity in the acid-insoluble fraction compriεing the DNA (Lehman, I.R., et al., J.Biol.Chem. (1958) 233-163, the diεclosure of which iε incorporated herein by reference) .
Example 10.
Purification of DNA-polymerase from Thermus ruber
10 g cell pellet in 20 ml buffer A (K-phoεphate - 10 mM pH- 7.4, EDTA - 0.1 mM, dithiotreitol - 2 mM) , containing 0.025 M NaCl waε εonicated during 20 min in an ice bath. After that the εuεpenεion waε centrifugated at 100.000 g, 1,5 h, the cell debriε waε diεcarded and ammonium sulphate was added to the supernatant to 50% saturation.
After centrifugation (20 min, 10.000 g) 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- celluloεe.
Proteinε were eluted by linear gradient NaCl (0.1-0.3 M NaCl) in buffer A. The fractionε containing DNA-poly era- se were combined, applied to a column (1.6 x 10 cm) with Blue-Sepharose, and DNA-polymeraεe waε eluted with linear gradient NaCl (0.1-1 M NaCl) in buffer A. Fractionε con¬ taining DNA-polymeraεe activity were combined and applied to a column (1.6 x 5 cm) with hydroxy apatite. Proteinε were eluted with linear gradient K-phosphate (0.01-0.5 M) and fractionε containing DNA-polymeraεe were combined and dialysed in buffer A, contained 0.2 M NaCl, 50% glycerol, 0.1 % Tritone X-100.
Enzyme waε teεted for specific activity, diluted to 5 units/μl and stored at -20°C.
Example 11.
Purification of pyrophoεphataεe from Thermus aquaticus and Thermus Ruber
50 g of blomaεε of Thermus aquaticus or Thermus Ruber were εuεpended in 100 ml of buffer A, containing 0.02 M Tris- HCl pH 7.8, 25 mM NaCl, 0.001 M EDTA, 2 mM DTT and when kept on ice, the suspension was disintegrated by ultrasoni- fication for 30 min.
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 waε waεhed with the same buffer, the proteins were eluted with 0.025 to 0.25 M NaCl gradient (the reεt of the components aε in the buffer A) .
In the eluate, the activity of inorganic pyrophoεphataεe (IP) waε aεεayed. The IP-containing fractionε were pooled, and applied to a 1, 6-diaminohexyl agaroεe column. Chromatography on this sorbent makes it posεible to ensu¬ re an effective purification of the inorganic py¬ rophosphatase owing to the fact that its affinity to this εorbent iε very high and it is eluted there from a con¬ centration of about 0,5 M. After elution, 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% - λ 100.
Example 12.
To aεεeεε the efficiency of uεing combinationε of enzymes from different sources, a 8-kb DNA fragment of the λ phage has been amplified (the results are shown in Fig. 5) .
Amplification was done by using the following enzyme com- binations:
1. 2.5 units DNA polymerase Thermus thermophilus (line
1)
2. 2.5 units DNA polymerase Thermus thermophilus + 0.25 units pyrophosphataεe Thermus thermophilus (line 2)
3. 2.5 units DNA polymerase Thermus thermophilus + 0.25 unitε pyrophoεphataεe Thermus aquaticus (line 3)
4. 2.5 unitε DNA polymeraεe Thermus termophilus + 0.25 units pyrophosphatase Thermus ruber (line 4) 5. 2.5 units DNA polymeraεe Thermus aquaticus (line
5)
6. 2.5 unitε DNA polymerase Thermus aquaticus + 0.25 units pyrophoεphataεe Thermus aquaticus (line 6)
7. 2.5 unitε DNA polymeraεe Thermus aquaticus + 0.25 unitε pyrophosphatase Thermus thermophilus .
8. 2.5 units DNA polymeraεe Thermus aquaticus + 0.25 unitε pyrophoεphataεe Thermus ruber (line 8)
9. 2.5 unitε DNA polymerase Thermus ruber (line 9)
10. 2.5 units DNA polymerase Thermus ruber + 0.25 unitε pyrophoεphataεe Thermus ruber (line 10)
11. 2.5 unitε DNA polymeraεe Thermus ruber + 0.25 units pyrophosphatase Thermus aquaticus (line 11) 12. 2.5 units DNA polymerase Thermus ruber + 0.25 unitε pyrophoεphataεe Thermus thermophilus (line 12)
The reaction mixture (total volume 50 microliterε) contain- ing 67 mM Tris-HCL, pH 8.8, 16.6 mM ammonium εulphate, 1.5 mM magneεium chloride, 0.01% (v/v) Tween-20, 0.25 mM of each dNTP, 1 mg of lambda DNA, 15 pmoleε of each primer, indicated amountε of enzymeε, was amplified for 25 cycles at temperature cycling conditionε: 95 °C- 1 min (firεt cycle - 2 min) , 56 °C - 1 min (firεt cycle - 2 min) , 72 °C - 2.5 min (first cycle - 10 min, second cycle - 5 min, last cycle - 7 min) . After amplification, 5 microliters of each reactionε were loaded on a 0.8 % agaroεe gel, containing 0.5 microgramε/ml of ethidium bromide. After electrophore- εiε, the gel waε photographed by UV.
The comparative resultε of teεting of different enzyme combinations in respect to their ability to amplify DNA- phage λ, show that DNA polymerases in any combination in the presence of pyrophosphataεe are considerably more effective. The resultε testify to the fact that the sub¬ ject of the preεent invention, i.e. thermophillic DNA polymerase combination with inorganic pyrophosphatase for the optimization of DNA synthesis in vitro, is a univerεal phenomenon and doeε not depend on the enzyme source.
Example 13.
We have also investigated the ability of thermostable pyrophosphatase from Thermus thermophilus to enchance efficiency of coupled reverse transcription/polymeraεe chain reaction (RT/PCR) performed by DNA polymerase from Thermus thermophilus in the preεence of CD-4 mRNA aε template.
RNA iεolation
Total cellular RNA haε been extracted from CD4 + U937 cell line by uεing guanidinium-phenol-chloroform (GPC) method of Chomczynεki and Sacchi (Analytical Biochemiεtry 162, 156- 159 (1987) . Cellε (5 x 106) were rinεed 3 times with cold phosphate-buffered εaline (PBS) by centrifugation at 1000 x g for 5 min at 4 °C. After washing the cells were lysed directly with 0.5 ml of εolution containing 4M guanidiniu thiocyanate, 25 M εodium citrate, pH 7; 0.5 % εarcosyl, 0.1 M 2-mercaptoethanol. Sequentially, 0.05 ml of 2M sodium acetate, pH 4, 0.5 ml of phenol (DEPC-water saturated) , and 0.1 ml of chloroform-isoamyl alcohol mixture (49:1) were added to the lysate and the phaseε mixed by vortexing briefly. The mixture waε kept on ice for 15-30 min and the phaεeε were separated by centrifuging at 12000 x g for 20 min at 4 °C. The upper aqueouε phase waε transferred to a new tube and an equal volume of isopropanol waε added. The sample was mixed and placed at -20 °C for 1 hr. RNA was collected by centrifugation (12000 x g, 20 min, 4 °C) . Pelleted RNA was washed twice with 70% ethanol, and the RNA was collected by centrifugation at 12000 x g for 10 min after each wash. The pellet was dried and resuspended in 100 μl of DEPC-treated H20. The RNA concentration waε estimated by UV absorbance at 260 nm.
Detection of CD4 mRNA in total RNA εa ple The potency of native Tth DNA polymerase to catalyse the coupled RT/PCR reactionε haε been already deεcribed by Glukhov et al. (ATB'92 conference. Advanced technology for clinical laboratory and Biotechnology, Abεtractε p.201 Milan, Italy, 1992) . We uεed in the experiment primers on mRNA of CD4-receptor protein flanking a 356-bp region. The sequences of primers were:
ARl-εense primer (position on mRNA CD-4 ;145-174) : 5'CAGGGAAACAAAGTGGTGCTGGGCAAA3 ' AR2-anti-εenεe primer (poεition on mRNA CD-4; 472- 501) : 5'GGTCAGGGTCAGGCTCTGCCCCTGAAGCAG3 ' . A coupled RT/PCR reaction was run aε deεcribed by Myers and Gelfand (Biochemistry 30, 7661-7666 (1991) . RT reaction (total volume 20 μl) containing 67 mM Tris-HCl pH 8.8, 16.6 mM (NH4)2S04, 1 mM MnCl2, 200 μM each of dATP, dCTP, dGTP, dTTP, 15 pmol primer anti-senεe (AR2) , 200 ng of total cellular RNA as template, 5 units of DNA polymerase from Thermus thermophilus and indicated amounts of pyropho- εphataseε from Thermus thermophilus or Thermus aquaticus . RT reaction mixtureε were overlaid with 70 μl of mineral oil and incubated at 70 °C for 15 min. After incubation the εamples were placed on ice.
PCR mixture (total volume 80 μl) containing 67 mM Triε- HC1, pH 8.8, 16.6 mM (NH4)2S04, 0.75 mM EDTA, 0.025 % (v/v)
Tween-20, 5 % (v/v) glycerol, 1.5 mM MgCl2, 15 pmol primer εense (AR1) was introduced beneath the oil to each RT reaction, followed by centrifugation for 30 seconds. The samples were amplified for 30 cycles using temperature cycling conditionε: 95 °C - 1 min (2 min for firεt cycle) ,
60 °C - 1 min (7 min for laεt cycle) . Aliquotε (10 μl) of each RT/PCR εamples were analyzed by electrophoresiε on a
1.5 % agaroεe gel containing 0.5 μl/ml of ethidium bromide.
After electrophoreεiε the gel was analyzed by using UV- transilluminator and photographed. Figure 6 shows the yield of specific 356-bp product after RT/PCR using the following enzyme combinations:
1. 5 unitε of DNA polymerase Thermus thermophilus . 2. 5 units of DNA polymeraεe Thrmus thermophilus + 0.05 unitε of pyrophoεphataεe Thermus thermophilus .
3. 5 unitε of DNA polymeraεe Thermus thermophilus + 0.1 units of pyrophoεphataεe Thermus thermophilus .
4. 5 unitε of DNA polymeraεe Thermus thermophilus + 0.25 unitε of pyrophoεphataεe Thrmus thermophilus .
5. Control - without RNA template.
6. 5 unitε of DNA polymerase Thermus thermophilus + 0.25 units of pyrophosphataεe Thermus aquaticus .
7. Control - RT reaction waε performed with εenεe primer (AR1) .
8. Control - human genomic DNA aε template. M. DNA molecular weight markers: φX 174 ds DNA digested Hae III restriction endonuclease.
Reεults of testing the ability of DNA polymerase Thermus thermophilus to reverse tranεcribe and amplify CD4 mRNA aε template εhow that DNA polymeraεe in the preεense of pyrophosphatase is conεiderable more effective. Moreover, this phenomenon does not depend on the pyrophosphataεe εource.