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of the desired polymerase should be taken into consideration. 5N/1S primers are recommended for Taq, whereas 3N/1S primers should be used with Vent and Pfu polymerases. Acknowledgments. We thank Dr. Ga´bor Ra´khely for his valuable help and Andrea Vo¨ro¨s for technical assistance.
REFERENCES 1. Ga´l, J., Schnell, R., Szekeres, S., and Ka´lma´n, M. (1999) Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR). Mol. Gen. Genet. 260, 569 –573. 2. Takeshita, M., Chang, C.-N., Johnson, F., Will, S., and Grollman, A. P. (1987) Oligodeoxynucleotides containing synthetic abasic sites. J. Biol. Chem. 262, 10171–10179. 3. Ng, L., Weiss, S. J., and Fisher, P. A. (1989) Recognition and binding of template-primers containing defined abasic sites by Drosophila DNA polymerase ␣ holoenzyme. J. Biol. Chem. 264, 13018 –13023. 4. Takeshita, M., and Eisenberg, W. (1994) Mechanism of mutation on DNA templates containing synthetic abasic sites: Study with a double strand vector. Nucleic Acids Res. 22, 1897–1902. 5. Paz-Elizur, T., Takeshita, M., Goodman, M., O’Donnell, M., and Livneh, Z. (1996) Mechanism of translesion DNA synthesis by DNA polymerase II. J. Biol. Chem. 271, 24662–24669. 6. Moran, S., Ren, R. X.-F., Sheils, C. J., Rumney, S., IV, and Kool, E. T. (1996) Non-hydrogen bonding ’terminator’ nucleosides increase the 3⬘-end homogeneity of enzymatic RNA and DNA synthesis. Nucleic Acids Res. 24, 2044 –2052. 7. Paz-Elizur, T., Takeshita, M., and Livneh, Z. (1997) Mechanism of bypass synthesis through an abasic site analog by DNA polymerase I. Biochemistry 36, 1766 –1773. 8. Efrati, E., Tocco, G., Eritja, R., Wilson, S. H., and Goodman, M. F. (1997) Abasic translesion synthesis by DNA polymerase ␤ violates the “A-rule”. J. Biol. Chem. 272, 2559 –2569. 9. Shibutani, S., Takeshita, M., and Grollman, A. P. (1997) Translesional synthesis on DNA templates containing a single abasic site. J. Biol. Chem. 272, 13916 –13922. 10. Shimizu, H., Yagi, R., Kimura, Y., Makino, K., Terato, H., Ohyama, Y., and Ide, H. (1997) Replication bypass and mutagenic effect of ␣-deoxyadenosine site-specifically incorporated into single-stranded vectors. Nucleic Acids Res. 25, 597– 603. 11. Greagg, M. A., Fogg, M. J., Panayotou, G., Evans, S. J., Connolly, B. A., and Pearl, L. H. (1999) A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil. Proc. Natl. Acad. Sci. USA 96, 9045–9050. 12. Horn, T., and Urdea, M. S. (1986) A chemical 5⬘-phosphorylation of oligodeoxyribonucleotides that can be monitored by trityl cation release. Tetrahedron Lett. 27, 4705– 4708. 13. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 14. Newton, C. R., Holland, D., Heptinstall, L. E., Hodgson, I., Edge, M. D., Markham, A. F., and McLean, M. J. (1993) The production of PCR products with 5⬘ single-stranded tails using primers that incorporate novel phosphoramidite intermediates. Nucleic Acids Res. 21, 1155–1162. 15. Chien, A., Edgar, D. B., and Trela, J. M. (1976) Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J. Bacteriol. 127, 1550 –1557. 16. Tindall, K. R., and Kunkel, T. A. (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27, 6008 – 6013.
17. Lundberg, K. S., Shoemaker, D. D., Adams, M. W. W., Short, J. M., Sorge, J. A., and Mathur, E. J. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1– 6. 18. Mattila, P., Korpela, J., Tenkanen, T., and Pitkanen, K. (1991) Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—An extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19, 4967– 4973. 19. Hu, G. (1993) DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3⬘ end of a DNA fragment. DNA Cell Biol. 12, 763–770. 20. Kunkel, T. A., Schaaper, R. M., and Loeb, L. A. (1983) Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro. Biochemistry 22, 2378 –2384. 21. Sagher, D., and Strauss, B. (1983) Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: Uniqueness of adenine nucleotides. Biochemistry 22, 4518 – 4526.
A Noncommercial Dual Luciferase Enzyme Assay System for Reporter Gene Analysis 1 Benjamin W. Dyer, Fernando A. Ferrer, Donna K. Klinedinst, and Ronald Rodriguez 2 Brady Urological Institute, Johns Hopkins Hospital, Marburg Room 145, 600 North Wolfe Street, Baltimore, Maryland 21287-2101 Received February 3, 2000
Key Words: reporter gene assay; Renilla luciferase; firefly luciferase.
The development of luciferase-based protocols for assessing reporter gene activity has enabled rapid and sensitive evaluations of promoter and enhancer constructs. To normalize for transfection efficiency, however, a second reporter gene is often used. Recent commercial strategies have been developed combining assessment of both the firefly (Photinus pyralis) luciferase enzyme and the Renilla (Renilla reniformis) luciferase enzyme sequentially within the same reaction tube. Unfortunately, since the components of these assay systems are proprietary, the assays can be quite costly. We described a dual luciferase reporter assay combining both the firefly luciferase enzyme and the Renilla luciferase enzyme in a nonproprietary buffer. 1 Funding sources: CapCure, Robert Wood Johnson Foundation, NIH SPORE CA58236, and NIH Training Grant 5-T32-DK-0755213. 2 To whom correspondence should be addressed. Fax: 410-9550833.
Analytical Biochemistry 282, 158 –161 (2000) doi:10.1006/abio.2000.4605 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS
FIG. 1. Comparison of the dynamic range of linear activity. Both the commercial and the noncommercial assays were carried out using a wide range of concentrations of purified recombinant luciferase.
Materials and Methods Cells and media. TSU-PR1 cells (a highly anaplastic human prostatic cancer cell line) was obtained as a generous gift of Jonathan W. Simons, Johns Hopkins Hospital. Transient transfection was performed with 10 g of the reporter Renilla luciferase plasmid pRLCMV (Promega, Inc.) into a single T25 flask of TSU cells at 80% confluence using Lipofectamine Plus (Life Technologies, Grand Island) as directed in their product insert. After 48 h the cells were lysed in 1 mL of
passive lysis buffer (Promega, Inc.) and stored at ⫺80°C. The cell lysate was then used for Renilla luciferase activity. Reagents and instrumentation. The luciferase assay buffer and Renilla buffer were prepared fresh for each experiment in this manuscript. Commercial dual luciferase (Promega) reagents were used as recommended by the manufacturer. The noncommercial stock luciferase assay buffer was made as follows: 25 mM glycylglycine, 15 mM K x PO 4 (pH 8.0), 4 mM EGTA, 2 mM ATP, 1 mM DTT, 3 15 mM MgSO 4, 0.1 mM CoA, 75 M luciferin, with the final pH adjusted to 8.0. The stock Renilla assay buffer was made as follows: 1.1 M NaCl, 2.2 mM Na 2EDTA, 0.22 M K x PO 4 (pH 5.1), 0.44 mg/mL BSA, 1.3 mM NaN 3, 1.43 M coelenterazine, with the final pH adjusted to 5.0. The estimated cost of performing 1000 assays using the noncommercial assay system described is about $20, compared to $900 for the commercial kit. Light emission was measured in a Wallac Lumat LB 9507 luminometer for the kinetic analysis and a Wallac 1450 Microbeta Jet liquid scintillation counter and luminescence reader for the 96-well microtiter plate assays. Measurement of luciferase activity. Serial dilutions of purified recombinant firefly luciferase enzyme (Promega) covering an 11-log range of concentrations were made in 10 mM Tris (pH 7.5) and 1 mg/ml. An aliquot 3
Abbreviations used: DTT, Dithiothreitol; BSA, bovine serum albumin; RLU, relative light units; CoA, Coenzyme A.
FIG. 2. Kinetics of chemiluminescence. Recombinant firefly luciferase was assayed using both the commercial system (A) and the noncommercial system (B) and luminosity (absolute counts per second) was plotted at 1-s intervals. After the curves were allowed to plateau, the activity was quenched by the addition of the Renilla assay buffer.
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(20 L) of each dilution was plated in triplicate in opaque white 96-well plates. One hundred microliters of luciferase buffer or the commercial buffer was injected into each well and the well was assayed for firefly luciferase activity. The Wallac 1450 Microbeta Jet liquid scintillation counter and luminescence reader was set for a 5-s delay followed by a 5-s reading of luminosity. The results plotted represent the average reading with standard error of the mean (Fig. 1). A kinetics assay was performed using the Lumat LB 9507 (single sample, dual-injection luminometer) since this apparatus was designed for continuous readings at 1-s intervals (Fig. 2). Twenty microliters of a 165 nM stock solution of recombinant firefly luciferase enzyme was placed in the reaction tube. One hundred microliters of either the luciferase buffer or the Promega luciferase assay reagent was injected into the tube and luminescence readings were taken every second. Dual assays with varying concentrations of firefly luciferase enzyme and a constant concentration of Renilla luciferase enzyme were conducted (Figs. 3A and 3B). Ten microliters of Renilla cell extract at a constant concentration and 10 l of varying concentrations of firefly luciferase enzyme (7.5 e–10 M, to 7.5 e–12 M) were placed in wells in a microtiter plate in triplicate. Results The dynamic range of the firefly luciferase assay using the commercial and noncommercial buffers were assessed by measuring luminescence as a function of recombinant firefly luciferase activity (Fig. 1). The linear range of activity for both assays exceeded 7 logs, consistent with previously established results for commercial buffers (Promega product insert). To assess the kinetics of activity, measurements were assessed at 1-s intervals for both the commercial and noncommercial buffers. Total luminosity of the noncommercial buffer was comparable to that of the commercial buffer (3.88 million relative light units [CPS] compared to 3.91 million CPS) (Figs. 2A and 2B). In the second phase of these experiments, the Renilla buffer was added to the reaction well to assess the adequacy and kinetics of quenching the firefly luciferase activity. As can be seen in Fig. 2, the commercial and noncommercial buffers almost completely quench the firefly luciferase activity within 2 s. In both cases the ability of the Renilla buffer to quench background firefly activity exceeded 99.9% of the starting activity. As a final assessment dual luciferase assays were performed in the same reaction well (Fig. 3). For these experiments a constant concentration of Renilla luciferase was placed in each well of the microtiter plates, along with varying concentrations of firefly luciferase. A zero concentration of firefly luciferase was also assayed. As expected, the measured luminescence de-
FIG. 3. Dual activity of firefly and Renilla luciferase. Varying concentrations of purified recombinant firefly luciferase were mixed with a fixed amount of Renilla luciferase (10 l of pRL-CMV transfected TSU-PR1 cell lysate) and a dual assay was performed utilizing both the commercial assay (A) and the noncommercial assay (B). The commercial kit did provide a nominally improved total firefly luciferase activity, with a significantly better Renilla luciferase activity, but at the expense of a higher background. Moreover, since the Renilla luciferase is a constitutive background marker for normalization purposes, its total activity can be adjusted by varying the concentration of the transfection with pRL-CMV. Quantitative measurement of activity was excellent with either assay system.
creased as the concentration of firefly luciferase in the reaction tube decreased, but the Renilla luciferase activity remained a constant. Discussion The firefly luciferase reaction is an ATP-dependent chemiluminescent reaction in which the enzyme lucif-
NOTES & TIPS
erase catalyzes the oxidative decarboxylation of its substrate (luciferin) producing oxyluciferin and light at a wavelength of 562 nm (1– 4). The reaction requires ATP, molecular oxygen, and a basic pH. The addition of Coenzyme A allows a more sustained plateau of activity facilitating subsequent analysis (5–7). By linking a promoter sequence to this easily detectable reporter gene, promoter activity can be measured quantitatively as a function of the light emitted in the luciferase assay. Unfortunately, various factors can affect total gene expression in transient transfections, complicating the analysis of promoter activity. Cotransfection of a second constitutively expressed reporter, followed by sequential measurement of both reporters in the same reaction tube, permits normalization (8). Currently, all of the commercially available dual luciferase kits are proprietary (Promega, Boehinger Mannheim) and expensive, and the contents of their buffers and substrates are unknown. Because we are involved in high throughput luciferase assays for a variety of promoters, we sought a cheaper, but comparable, alternative. In summary, we report a noncommercial dual luciferase assay system that performs comparably to commercially available kits. Our assay system allows detection of luciferase activity over a 7-log range of concentrations. The kinetics of activation and quenching are nominally less than those of the commercial equivalent, but still allow excellent detection as long as the detector is programmed to allow a sufficient lag time for maximal activity (5 s). Most importantly, however, this fully defined protocol allows investigators the cost-effective option of preparing their own reagents for dual luciferase assays. REFERENCES 1. Bronstein, I., Fortin, J., Stanley, P. E., Stewart, G. S., and Kricka, L. J. (1994) Chemiluminescent and bioluminescent reporter gene assays. Anal. Biochem. 219, 169 –181. 2. Mittal, S. K., Bett, A. J., Prevec, L., and Graham, F. L. (1995) Foreign gene expression by human adenovirus type 5-based vectors studied using firefly luciferase and bacterial beta-galactosidase genes as reporters. Virology 210, 226 –230. 3. Iizumi, T., Yazaki, T., Kanoh, S., Kondo, I., and Koiso, K. (1987) Establishment of a new prostatic carcinoma cell line (TSU-Pr1). J. Urol. 137, 1304 –1306. 4. Bronstein, I., Martin, C. S., Fortin, J. J., Olesen, C. E., and Voyta, J. C. (1996) Chemiluminescence: Sensitive detection technology for reporter gene assays. Clin. Chem. 42, 1542–1546. 5. Steghens, J. P., Min, K. L., and Bernengo, J. C. (1998) Firefly luciferase has two nucleotide binding sites: Effect of nucleoside monophosphate and CoA on the light-emission spectra. Biochem. J. 336, 109 –113. 6. Ford, S. R., Hall, M. S., and Leach, F. R. (1992) Enhancement of firefly luciferase activity by cytidine nucleotides. Anal. Biochem. 204, 283–291. 7. Ford, S. R., Buck, L. M., and Leach, F. R. (1995) Does the sulfhydryl or the adenine moiety of CoA enhance firefly luciferase activity? Biochim. Biophys. Acta 1252, 180 –184.
8. Stables, J., Scott, S., Brown, S., et al. (1999) Development of a dual glow-signal firefly and Renilla luciferase assay reagent for the analysis of G-protein coupled receptor signalling. J. Recept. Signal Transduct. Res. 19, 395– 410.
Reaction of Tris(2-carboxyethyl)phosphine (TCEP) with Maleimide and ␣-Haloacyl Groups: Anomalous Elution of TCEP by Gel Filtration Douglas E. Shafer,* John K. Inman,† and Andrew Lees* ,‡ ,1 *Virion Systems, Inc. and ‡Biosynexus, Inc., 9610 Medical Center Drive, Rockville, Maryland 20850; and †Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received March 21, 2000
Thiolated peptides are frequently used in the preparation of peptide conjugates, especially in the preparation of immunogens. Usually, synthetic thiol peptides are partially oxidized to disulfides and must be reduced before being coupled to a carrier-bearing electrophilic groups. Dithiothreitol (DTT) 2 is commonly employed to reduce peptide disulfides for this purpose, but this reagent must be removed from the solution, usually by gel filtration, before the reduced peptide can be used. However, low molecular weight (e.g., ⬍1500 Da) peptides may be included in the gel filtration matrix and therefore not easily separated by gel filtration from the DTT, since the latter is often used in large excess. Therefore, alternative means (e.g., ion-exchange) may have to be employed to isolate the reduced peptide. Trialkylphosphines are powerful and selective reductants for disulfides (1), but until recently, they have not been commonly used in the life sciences due to their being malodorous and/or water insoluble (2). The commercial availability of tris-(2-carboxyethyl)phosphine hydrochloride (TCEP), which is odorless and water soluble, makes this reagent safe and convenient to use ((2), Pierce Chemical Co., Molecular Probes). TCEP rapidly and stoichiometrically reduces most peptide or other disulfides, even under acidic conditions (2). Furthermore, aqueous solutions of TCEP are reasonably stable (2, 3), and the functional concentration of TCEP 1 To whom correspondence should be addressed. E-mail: [email protected]
2 Abbreviations used: DTT, dithiothreitol; TCEP, tris-(2-carboxyethyl)phosphine hydrochloride; IAA, iodoacetamide; NEM, N-ethylmaleimide; 4-DTP, 4,4⬘-dithiopyridine; 2-ME, 2-mercaptoethanol; BSA, bovine serum albumin.
Analytical Biochemistry 282, 161–164 (2000) doi:10.1006/abio.2000.4609 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.