We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP—Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTP

Krieg, Alexander

Laib, Stephan

Ruckstuhl, Thomas

Seeger, Stefan

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P1: KEFJournal of Fluorescence [JOFL] pp1125-jofl-478622 January 21, 2004 22:48 Style file version 29 Aug, 2003Journal of Fluorescence, Vol. 14, No. 1, January 2004 (©2004)Real-Time Detection of Polymerase Activity UsingSupercritical Angle FluorescenceAlexander Krieg,1 Thomas Ruckstuhl,1 Stephan Laib,1 and Stefan Seeger1;2Received September 9, 2003; accepted September 30, 2003We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with poly-merases by complementary strand synthesis. For this reason single stranded DNA was immobilizedon a coverslip and the increase of fluorescence due to the synthesis of the corresponding strandwith tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. Bycomparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP—Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymesand dNTPs.KEY WORDS: Biosensors; fluorescence dyes; polymerase; primer elongation.INTRODUCTIONPolymerases are of great importance for multiply-ing DNA in vivo as well as in vitro. Enzymatic doublestrand synthesis is used in many different applicationsof modern biology, such as polymerase chain reaction(PCR) [1], DNA sequencing [2–5] or gene expression[6]. Evolution has created a large variety of polymeraseswith different features [7]. One prominent example is theTaq-polymerase with working temperatures up to 80–C,which plays the fundamental role in PCR.To sequence DNA with fluorescent methods enzymesare desired, that are able to synthesize double strandedDNA (dsDNA) containing dye labelled bases. Due to sterichindrances the incorporation of dye-dNTPs turned out tobe quite inefficient with most polymerases. In any casethe enzymatic activity is affected by the label, neverthe-less some species have been found to deliver reasonableincorporation yields under certain conditions [8,9].In the present paper we studied surface bound dsDNAsynthesis in order to optimize the incorporation efficiency1 Physikalisch-Chemisches Institut, Universita¨t Zu¨rich, Winterthurerstr.190, 8057 Zu¨rich, Switzerland.2 To whom correspondence should be addressed. E-mail: sseeger@pci.unizh.chof Cy5 fluorophores. We used three enzymes known tocope with the markers, T7-polymerase, Klenow-fragmentand Cyscribe reverse transcriptase, varied the reactiontemperature and compared the yield of both commerciallyavailable Cy5-dNTPs, i.e. Cy5-dCTP and Cy5-dUTP(ribonucleic acid pendant of dTTP).The incorporation of Cy5 into the surface boundcomplexes was detected by measuring the supercriticalangle fluorescence (SAF) emission in real time with abiosensor of custom design [10].MATERIALS AND METHODSPreparation of poly-L-lysine (PLL) coatedcoverslips: The substrates (Menzel-Glaser, Germany)were cleaned in an ultrasonic bath in ethanol solu-tion (70%, 30 min), in aqueous NaOH/ethanol (25 gNaOH in 250 mL 60% ethanol, 2 hr) and rinsed withbidistilled water. Subsequently they were prepared ina PLL-solution (0.01% w/v, Sigma Diagnostics, USAin PBS buffer, pH 7.48 Fluka, Switzerland ) for 1 hrand dried at 45–C in a vacuum oven. Then they weremodified in glutaraldehyde solution (20 mL, 2.5%Fluka, Switzerland) for 1 hr and rinsed with bidistilledwater.751053-0509/04/0100-0075/0 C° 2004 Plenum Publishing CorporationP1: KEFJournal of Fluorescence [JOFL] pp1125-jofl-478622 January 21, 2004 22:48 Style file version 29 Aug, 200376 Krieg, Ruckstuhl, Laib, and SeegerPrimer Annealing and Couplingof Aminated ssDNA to the GlassFor the comparison of the incorporation efficienciesof the enzymes 100 nucleotides (nt) long ssDNA contain-ing 15 guanine bases was chosen, which provides incor-poration for 15 Cy5 tagged dCTPs, as for the comparisonof the incorporation of the two bases (dCTP and dUTP)55 nt long ssDNA containing three guanine respectivelythree adenine bases were in use (ssDNA purchased byMicrosynth Switzerland). After primer annealing to the30end the ssDNA was coupled with its amine labelled 50endto the surface [11].Preparation of DNA SynthesisAfter gluing the coverslips to a mask containing sixreaction vessels a mixture of dNTPs (unlabelled 3:3£10¡6 M, MBI Fermentas, Germany and 3:3£ 10¡7 MCy5-dCTP or Cy5-dUTP, Amersham Biosciences) andone unit of enzyme (Klenow fragment or Sequenase T7polymerase, Cyscribe reverse transcriptase, AmershamBiosciences, unit defined by supplier) was dissolved in re-action buffer (purchased with the specific enzymes). Afteradding the dNTP mix the reaction was started by pipettingthe enzyme.Fluorescence DetectionIn order to detect the incorporation of Cy5 into sur-face bound DNA in presence of high dye concentrations itis necessary to restrict the fluorescence detection volumeto the interface as much as possible. We have developed anoptical biosensor that detects the supercritical angle fluo-rescence (SAF), reducing the penetration of the detectionvolume into the solution to less than 100 nm [12,13]. Tovary the temperature of the experiments we used a home-made heating control.RESULTSThe Cyscribe enzyme, which is a mutated MML-Vreverse transcriptase, is in use to transcribe RNA intocDNA and integrate Cy5-dCTP. MML-V reverse tran-scriptase is known to feature polymerase activity. How-ever we found, that dsDNA synthesis at the interface withCyscribe does not take place at all, as we detected noincrease of surface fluorescence. The exonuclease freeKlenow fragment is widely used in biochemical appli-cations for its high reliability. It has been shown that it isable to incorporate dyes into DNA [9]. The more recentlydeveloped T7 polymerase was claimed to be more efficientfor the incorporation of dye-dNTPs.To investigate the influence of the temperature onpolymerase activity we measured the synthesis with theKlenow fragment at 25–C and 38–C and with the T7 poly-merase at 25–C and 41–C. Each reaction was repeatedthree times. The averaged fluorescence increases duringdsDNA synthesis with Klenow and T7 are shown in Fig. 1aand b respectively. In all shown figures the backgroundsignal was subtracted from the data and the start time wasset to zero. In order to minimize photobleaching the in-tensity was gathered every 90 s, the rest of the time thelaser was blocked by means of a mechanical shutter. Thevelocities of the reactions are rather slow, as the rise ofFig. 1. a: Fluorescence signal vs. time during DNA strand synthesis.Reaction temperature comparison of T7 polymerases. Squares: Averagemeasurement with T7 polymerase at 42–C; Circle: Average measurementwith T7 polymerase at room temperature. b: Fluorescence signal vs.time during DNA strand synthesis. Reaction temperature comparison ofKlenow fragment (exonuclease free). Circle: single measurement withKlenow fragment at 38–C; squares: Average measurement with Klenowfragment at room temperature.P1: KEFJournal of Fluorescence [JOFL] pp1125-jofl-478622 January 21, 2004 22:48 Style file version 29 Aug, 2003Real-Time Detection of Polymerase Activity 77the fluorescence signal was not completed within 30 min.Compared to their natural working conditions the situa-tion for the enzymes is complicated by steric hindrancesby the dye and the surface. Varying the temperature hassignificant influence upon the reaction kinetics. For bothpolymerases a higher activity was measured at room tem-perature. The Klenow fragment showed a higher reactionvelocity at 25–C, the yield of the product however wasfound to be nearly independent from temperature. Thereaction yield as well as velocity of T7 polymerase isstrongly decreased at increased the temperature. The ex-planation for this effect is, that the enzymes have a broadtemperature intervall in which they are effective, but theyare more stable at room temperature. Also enzymes de-grade faster at higher temperature, so there exists anoverlay of two processes.A comparison of the two polymerases at 25–C isgiven at Fig. 2. Accordingly the Klenow fragment is moreefficient in building up labelled dsDNA at the interface.Hence we used the Klenow fragment to study the influenceof exchanging the dye labelled base. To incorporate Cy5-dUTP we immobilized ssDNA containing three adeninesand for Cy5-dCTP incorporation ssDNA containing threeguanines. As shown in Fig. 3 the enzyme is bearly able tocope with the labelled uracile bases.DISCUSSIONTo date no polymerase/dye-dNTP combination isknown, that approaches the incorporation rates of the en-Fig. 2. Fluorescence signal vs. time during DNA strand synthesis at roomtemperature. Comparison of Klenow fragment and T7 polymerases.Triangles: Average of the measurement with Klenow (exonuclease free)fragment; circles: Average of the measurement with T7 polymerase.Fig. 3. Fluorescence signal vs. time during DNA strand synthesis. Com-parison of dCTPs and dUTPs Circles: Average incorporation of 3 Cy5-dCTPs; triangles: Average incorporation of 3 Cy5-dUTPs.zymes under natural conditions. For this reason the searchand synthesis for polymerase—dye tagged dNTP systemsis going on for maximizing the yield of incorporated la-belled dNTPs and conclusively fast and reliable methodsfor proving the efficiency are essential [14,15].The collected data suggest that the best of the inves-tigated dye-enzyme pairs for an accurate incorporationefficiency is the Klenow-fragment—Cy5-dCTP. The ad-vantage of the developed biosensor lies in the easy andcontinuous collecting of the data. There is no need forfurther interference after adding of the reaction mixtureand the kinetics of the whole reaction are measured andnot just the endpoint signals. Besides measurement of thepolymerase activity this setup also allows detection ofSingle Nucleotide Polymorphisms (SNPs) based i. e. onprimer elongation. The next step for this setup is to reducethe background fluorescence by blocking the surface andincreasing the quantum yield per incorporated dye [16,17].REFERENCES1. K. Mullis, F. Faloona, S. Scharf, R. Saiki, G. Horn, and H. Erlich(1986). Specific enzymatic amplification of DNA invitro—The poly-merase chain-reaction. Cold Spring Harb. Sym. 51, 263–273.2. F. Sanger, S. Nicklen, and A. R. Coulson (1977). DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A.74(12), 5463–5467.3. E. D. Hyman (1988). A new method of sequencing DNA. Anal.Biochem. 174(2), 423–436.4. M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H. G. Craighead,and W. W. Webb (2003). Zero-mode waveguides for single-moleculeanalysis at high concentrations. Science 299(5607), 682–686.5. L. T. C. Franca, E. Carrilho, and T. B. L. Kist (2002). A review ofDNA sequencing techniques. Q. Rev. Biophys. 35(2), 169–200.P1: KEFJournal of Fluorescence [JOFL] pp1125-jofl-478622 January 21, 2004 22:48 Style file version 29 Aug, 200378 Krieg, Ruckstuhl, Laib, and Seeger6. M. Schena, R. A. Heller, T. P. Theriault, K. Konrad, E. Lachenmeier,and R. W. Davis (1998). Microarrays: Biotechnology’s discoveryplatform for functional genomics. Trends Biotechnol. 16(7), 301–306.7. S. Brakmann (2001). Discovery of superior enzymes bydirected molecular evolution. Chembiochem. 2(12), 865–871.8. H. Tveit and T. Kristensen (2001). Fluorescence-based DNA poly-merase assay Anal. Biochem. 289(1), 96–98.9. S. Brakmann and P. Nieckchen (2001). The large fragment of Es-cherichia coli DNA polymerase I can synthesize DNA exclusivelyfrom fluorescently labeled nucleotides. Chembiochem. 2(10), 773–777.10. A. Krieg, S. Laib, T. Ruckstuhl, and S. Seeger (2003).Real-time detection of nucleotide incorporation during com-plementary DNA strand synthesis. Chembiochem. 4(7), 589–592.11. N. Zammatteo, L. Jeanmart, S. Hamels, S. Courtois, P. Louette,L. Hevesi, and J. Remacle (2000). Comparison between differentstrategies of covalent attachment of DNA to glass surfaces to buildDNA microarrays. Anal. Biochem. 280(1), 143–150.12. T. Ruckstuhl, M. Rankl, and S. Seeger (2003). Highly sensitivebiosensing using a supercritical angle fluorescence (SAF) instru-ment. Biosens. Bioelectron. 18(9), 1193–1199.13. J. Enderlein, T. Ruckstuhl, and S. Seeger (1999). Highly efficient op-tical detection of surface-generated fluorescence. Appl. Optics 38(4),724–732.14. G. Giller, T. Tasara, B. Angerer, K. Muhlegger, M. Amacker,and H. Winter (2003). Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthe-sis of various reporter group-labeled 20-deoxyribonucleoside-50-triphosphates. Nucl. Acids Res. 31(10), 2630–2635.15. T. Tasara, B. Angerer, M. Damond, H. Winter, S. Dorhofer, U.Hubscher, and M. Amacker (2003). Incorporation of reportermolecule-labeled nucleotides by DNA polymerases. II. High-densitylabeling of natural DNA. Nucl. Acids Res. 31(10), 2636–2646.16. T. Scott, S. Smith, B. Windle, and A. Guiseppi-Elie (2003). Impactof surface chemistry and blocking strategies on DNA microarrays.Nucl. Acids Res. 31(16), e87.17. J. Malicka, I. Gryczynski, B. P. Maliwal, J. Y. Fang, and J. R.Lakowicz (2003). Fluorescence spectral properties of cyanine dye la-beled DNA near metallic silver particles. Biopolymers 72(2), 96–104.

Real-Time Detection of Polymerase Activity Using Supercritical Angle Fluorescence

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Real-Time Detection of Polymerase Activity Using Supercritical Angle Fluorescence

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