Anal. Chem. 1992, 64, 1996-1997
1896
CORRESPONDENCE
Real-Time Monitoring of DNA Polymerase Reactions by a Micro ISFET pH Sensor Toshinari Sakurait and Yuzuru Husimi' Department of Environmental Chemistry, Saitama University, Urawa 338, Japan Real-time monitoring of enzymatic DNA (or RNA) polymerization reactions is required in the various fields of biotechnology, especially in diagnostic tests based on DNA polymerization and in evolutionary molecular engineering.'P2 In the latter field, one of the key processes is a selection of rapidly growing DNA (or RNA) clones, and the real-time monitoring has been realized through optical methods including fluorescence intensity measurement from a dye molecule bound to the polynucleotide.3 However, influences of the dye molecule on the reaction cannot be eliminated and the extension of the optical method to large-scale parallel monitoring will be difficult. Other characteristics required for the monitoring devices are as follows: a small size to correspond with the small amount of sample solutions, a fast response time, durability, and thermal resistance (for polymerase chain reaction (PCR)). Here we report on an electrochemical method using a miniaturized pH sensor made of ion-sensitive field effect transistors (ISFETs), which will satisfy almost all these characteristics. The ISFET, which has a gate insulator made of silicon nitride, is a fast transducer of hydrogen ion concentration into~oltage.~ A commercial ISFET pH meter is now available (e.g. pH Boy-C1, Shindengen Electric Manufacturing Co., Saitama). Its reference electrode, however, is made of gelimmersed Ag/AgCl, and it does not have some of the above mentioned characteristics. The unit step in the chain-elongation reaction by DNA polymerase can be written as
DNA
+ [dNTP] +
XH'
-
0-
I DNA-0-P-0-dN II 0
+
{PPi)
where {dNTP} and (PPi} are sets of various equilibrated ionization states for Mg complexes of deoxynucleotide triphosphates and for Mg complexes of inorganic pyrophosphate, respectively. The stoichiometric parameter x is the effective (fractional) number of H+ uptake when one nucleotide is incorporated into the 3' end of the DNA strand. For each pH value, x can be calculated from the pK values of the various ionization groups in the Mg complexes of dNTPs, pyrophosphate, and the phosphate moiety of DNA. To estimate x roughly, we used the pK value of ATP-Mg instead of dNTPMg. The calculation showed that x = +0.3 at pH 7 , that is,
* To whom correspondence should be addressed. + Present address: Bio-Medical Center, Hitachi Ltd., Ichige, KatsutaCity 312, Japan. (1) Eigen, M.; Gardiner, W. Pure A p p l . Chem. 1984, 56, 967. (2) Husimi, Y. Adu. Biophys. 1989, 25, 1. (3) Bauer, G . J.; McCaskill, J. S.; Otten, H. Proc. Natl. Acad. Sci. U.S.A. 1989,86, 7937. (4) Matsuo, T.; Wise, K. W. IEEE Trans. 1974, BME-21, 485. ( 5 ) Nishigaki, K.; Kaneko, Y.; Wakuda, H.; Husimi, Y.; Tanaka, T. Nucleic Acids Res. 1985, 13, 5747.
0003-2700/92/03641996$03.00/0
the chain-elongation reaction is accompanied by the H+ uptake at neutral pHe6 Therefore, if the buffer capacity is low, the pH sensor can monitor the time course of the chainelongation reaction by the increase in pH. The change in pH is a smooth but complex function of the extent of nucleotide incorporation, because both the parameter x and the buffer capacity are dependent on pH. A linear approximation, however, is not bad for semiquantitative use under our experimental conditions. EXPERIMENTAL SECTION pH Sensor. We constructed a pH sensor from two ISFETs (integrated with a temperature-sensor diode; width = 1mm; gift from Shindengen Co.) and a thin Pt wire as the reference electrode. Fluctuations originating from instability of the Pt electrode canceled out by rejecting the voltage common to the two ISFETs. The gate surface of one ISFET was processed with methyltrimethoxysilane vapor at 120 "C for 5 h in order to replace the ionization groups with the siloxane. This processing reduced the hydrogen ion sensitivity from -55 to -8 mV/pH at 37 "C.The other ISFET was not processed. The differential gate output voltage was recorded in a personal computer via a pair of constant drain-current preamplifiers (handmade), a difference amplifier (handmade), and a digital multimeter (HP3478A, YokogawaHewlett-Packard). The pH sensor required a sample solution of only 20 FL in an Eppendorf tube and had a response time (95% ) of within 2 s. The pH sensitivity was -46 and -50 mV/pH at 45 and 93 "C, respectively. As it had no temperature hysteresis, the temperature dependence was calibrated through the integrated diode. The pH sensor was not affected by Na+. It was not damaged by a dilute phosphate solution. DNA Polymerase Reaction. The Klenow fragment of Escherichia coli DNA polymerase I was purchased from Takara Shuzo Co. (Lot No. 1031). The template was a single-stranded (9s) circular DNA of phage M13 prepared in our laboratory. The primer (12-mer)was synthesized using aDNA synthesizer (Model 381A, Applied Biosystems Inc.). The weak buffer solution (initially pH 7.1) was 0.7 mM Tris-HC1,26.3 mM NaCl, 0.1 mM EDTA-Na2, 7.0 mM MgCl2 and contained 330 pmol/pL dNTP. The template and the primer were mixed at a mole ratio of 1:1, incubated in the buffer solution at 60 "C for 20 min, and annealed at 45 "C for 30 min. DNA polymerase was mixed into the preincubated substrate solution 5 min after immersion of the sensor. Restriction Fragment Analysis by Gel Electrophoresis. Every 5 min, a small portion was drawn from the solution of the DNA polymerase reaction. After deactivation of the polymerase at 93 "C for 10 min, DNA was digested with the restriction enzyme HaeIII (purchased from Takara Shuzo Co., Lot No. 505) at 37 O C for 30 min in the standard buffer, and the digest was loaded onto a 4% polyacrylamide gel with 8 M urea. After the electrophoresis, the bands were stained with silver and photographed. RESULTS AND DISCUSSIONS Figure l a shows a reaction curve monitored with the micro pH sensor a t 45.0 "C. At time zero, the polymerase (final (6) Sakurai,T.; Ootsuka, H.; Kondo, S.; Husimi, Y. Unpublished work. (Reported in Master Thesis by T. Sakurai, Saitama University, 1991.) @ 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64.NO. 17. SEPTEMBER 1. 1992
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Flg~182. Illustratkm of a part of the M13 saDNA template and a growing replica strand cm n. "D" denotes cunlng snes by HsslII cm dOuble-Stranded DNA, whereas " S denotes tb snes cut easlly by HeeIll even on ssDNA.
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Flgura 1. (a)TlmecourseofDNApolymerizationreactlononmeM13 slngbstranded(%%)DNAtemplate. catalyzed wkh the Klenow fragment as measured by tb micro ISFET pH sensor. The absclsss Is the time anermixlng wlththeenzymesolutlon. (b)Restrktkmfragmentanalysis of the Same reactlon mlxture by Heelll. The top and ttm bonom reglons of ths gel were cutoff for simplicity. Arrows link the sampllng time wkh the gel panern. See text.
concentration of 0.04 unit/pL) solution was mixed with a preincubated solution of the other reactants including the template M13 ssDNA (33fmol-polymer/pL) and the primer (lbmer, 33 fmol of oligomer/pL). Figure l h shows the result from a time-consuming electrophoretic analysis of the same reaction mixture as Figure la. 'PI" denotes the primer indicator (234-mer).that is, the single-stranded fragment cut at the first restriction site no. 1397 (223 hp downstream from the primer; see Figure 2) of the extended primer template. Between the site a t no. 1397 and the site at no. 5868, the Hoe111 cuts out two large fragments (not shown in Figure l h ) from both ssDNA and the extended primer template. 'H" denotes the H fragment the farcutting (142-mer)ofM13-DNAcuthyHae111,ofwhich site at no. 5726 is 2300 hp downstream from the primer. The H fragment isvery difficult tocut out ofthessDNA suhstrate.5 'IJ" denotes the fused I + J fragment (175-mer), which is eaaily cut out of the ssDNA. I t is difficult to cut ssDNA a t
site no. 5346 (2680 hp downstream) to make the I and the J.6 A t about 2450 hp downstream from the primer, between the H and the IJ region, there is a very stable hairpin structure known as the morphogenesis origin. There the polymerization is apt to stop, as indicated by a plateau in the reaction curve and also by the existence of an IJ hand even after 25 min. The change in the hand pattern (Figure l h ) is consistent with the reaction curve (Figure la). The hiphasic reaction curve (Firmre - la) mavindicateaslowine-downeffectofhahim structures near the H region. The same slowly rising curve was observed in repeated experiments. A t nonoptimum pH (6.1), a more slowly rising curve was obtained. For a reaction mixture without the template, the curve was not observed. The possibility of drift originating from the adsorption of the proteinor DNAtothegatesurface wasprovedto henegligihle. Artifacts originatingfrom pyrophosphate hydrolysis were ala0 negligihle.6 This micro pH sensor can be applied also to the monitoring of RNA polymerization reactions. After more extensive data processing (e.g., correction hy pH dependence of the buffer capacity and of the x-value, using pK data of dNTPs under our experimental condition, ete.), this sensor system may be applicable tomorequantitativestudyon the kineticsofDNA (or RNA) polymerase reactions using a very small amount of sample solution.
-
ACKNOWLEDGMENT ThisworkhasbeensupportedhyagrantfromtheMinistry of Education, Science, and Culture, Japan.
RECEIVED for review October 29, 1991. Accepted April 28, 1992. Registry No. DNA polymeraae, 9012-90-2.
