- Email: [email protected]
Generation of Multiple Site-Specific Mutations in a Single Polymerase Chain Reaction Product
288
NOTES & TIPS
under the same assay conditions) the activities at 30 and 40°C. Assay with the eluted protein at 50°C showed the presence of 75.96 units of glucoamylase and 0.72 units a-D-glucosidase bound to the immobilized antibody. Since the bound enzyme could be eluted from the antibody at 50°C and the eluted enzymes were pure as seen by SDS–PAGE after silver staining of the gels (data not shown), the procedure could be applied for activity determinations at higher temperatures. These experiments showed a temperature optima at 60°C for glucoamylase and a-D-glucosidase (Fig. 2) defining applicability of the procedure for thermostability determinations of enzymes. The procedure avoided harsh treatments generally used for elution procedures (11). The elution is apparently due to temperature and can be employed with enzymes stable at higher temperatures. The temperature optima described of these enzymes were significantly different from the assay carried out with crude culture filtrates (Fig. 2). The interfering starch hydrolyzing enzymes contributed to increased temperature activities because we could purify an a-amylase active at acidic pH from the culture fluids and a-D-glucosidase showed activities on starch and dextrins but with different substrate specificities as defined by Km (10). Though the procedure involves an antibody, for enzyme-engineering studies, a single antibody preparation can be used to selectively isolate clones with improved activities at high temperature without interferences by other substances in the crude broth. Acknowledgments. The authors thank the director of the institute for facilities. This research was supported by the Council of Scientific and Industrial Research, New Delhi, through Junior Research Fellowships to C.S., A.K.D., and R.K.
REFERENCES 1. Frandsen, P. T., Christensen, T., Stoffer, B., Lehmbeck, J., Dupont, C., Honzatko, R. B., and Svensson, B. (1995) Biochemistry 34, 10162–10169. 2. Christensen, U., Olsen, K., Stofer, B. B., and Svennson, B. (1996) Biochemistry 35, 15009 –15018. 3. Spector, T. (1978) Anal. Biochem. 86, 142–146. 4. Shankar, C. S., and Umesh-Kumar, S. (1994) Microbiology 140, 1097–1101. 5. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. 6. Laemmli, U. K. (1970) Nature 227, 680 – 685. 7. Batteiger, B., Newhall, W. J., and Jones, R. B. (1982) J. Immunol. Methods 55, 297–307. 8. Dahlqvist, A. (1961) Biochem. J. 80, 547–551. 9. Boel, E., Hjort, I., Svensson, B., Norris, F., Norris, K. E., and Fill, N. P. (1984) EMBO J. 3, 1097–1102. 10. Kita, A., Matsui, H., Samoto, A., Kimura, A., Takata, M., and Chiba, S. (1991) Agric. Biol. Chem. 55, 2327–2335. 11. Thomas, C. T., and McNamee, M. G. (1990) Methods Enzymol. 182, 499 –515.
Generation of Multiple Site-Specific Mutations in a Single Polymerase Chain Reaction Product Amom Ruhikanta Meetei and M. R. S. Rao Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Received May 13, 1998
Site-directed mutagenesis has been very valuable in studying the structure–function relationship of proteins (1). PCR-mediated mutagenesis has been the method of choice in recent years to change the desired amino acid from one to another. Several procedures have been described to achieve the desired mutation using PCR technology (2–13). Although in most of the cases the desired change is only with respect to one amino acid, sometimes one needs to generate several changes in the amino acid sequence to understand the biological function of a given protein. Such a necessity is apparent when one is dealing with a zinc finger class of proteins wherein multiple residues of cysteine and histidine are involved in the coordination of zinc. We have been working over the past several years on the structure–function relationship of a spermatidal specific protein TP2 which appears transiently during mammalian spermiogenesis. We have shown that this basic nuclear protein binds two atoms of zinc involving both cysteine and histidine residues (14) and this zincinduced polypeptide fold is necessary for interaction and condensation of DNA with a preference to alternating GC containing DNA (15). Furthermore, this observation led to our study demonstrating that TP2, in the zinc coordinated form, recognizes a human CpG island sequence (16). The amino acid sequences of rat, mouse, and human TP2 contain several cysteine and histidine residues in the N-terminal two-thirds of the molecule, more than what is required for coordination of two zinc atoms. Amino acid sequence alignment of the zinc binding domain of TP2 revealed that it does not have strong similarity with any of the known classes of zinc finger proteins. This has posed a challenge to identify the actual residues involved in zinc coordination. We have recently cloned and expressed rat TP2 in Escherichia coli which has paved the way for answering this question (17, 18). During this course of investigation, we had to develop multiple mutants in the same molecule. There are at least three reports describing methods for generating the multiple sitespecific mutations (19 –21). The first method described by Dwivedi et al. (19) is termed as the MM-SSP method in which double mutations were created by using the previous single-mutant clone as starting template and so forth. This method involves confirmation of each ANALYTICAL BIOCHEMISTRY 264, 288 –291 (1998) ARTICLE NO. AB982866
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS
FIG. 1. A schematic representation of the strategy for generating multiple site-specific mutations. S1, side primer; P1, P2, . . . , P6, mutagenic PCR products; m1, m2, . . . , m6, mutagenic primers; and S2, 39 end specific primer.
mutant clone every time before starting the next mutation. In the second method described by Tu and Sun (20), different mutagenic primers were used at a time to create a combination of mutations in a given gene. However, this method also involved generation of single-stranded DNA template containing dUMP and subsequent screening of mutant clones (essentially an extension of Kunkel’s method). Furthermore, the efficiency of mutagenesis was not very high. Recently, CCR (combined chain reaction) was developed by Bi and Stambrook (21) for generating single and multiple mutations. In this method, one needs special considerations, such as annealing efficiency of mutant primers should not be less than that of side primers and DNA polymerase should not have 59 to 39 exonuclease activity. Although this method appears highly efficient to generate single and double mutations, the efficiency of generation of three or more multiple mutations needs to be tested. In all three methods there is an absolute
289
requirement of DNA ligase (thermostable ligase in the third method) and 59 phosphorylation of mutagenic primers. We report here a simple and very efficient method to generate multiple site-specific mutations in a gene of interest wherein no DNA ligase, no 59 phosphorylation of mutagenic primers, no consideration of annealing efficiency of mutagenic primers compared with side primers in the same PCR condition, no extra care for 59 to 39 exonuclease activity of thermostable DNA polymerase, and no subcloning of PCR product each time the mutagenic primer is used are required. This method has also allowed us to create only the desired multiple mutations and not a combination of multiple mutations. One of the important requirements of the present method is to have two templates (A and B) in which one of the templates has an extra flanking sequence in addition to the gene or part of the gene of interest, to which one of the side primers (S1) binds while the other template has no binding site for this side primer. The second template can either have a different flanking side, as generated from two different vector backgrounds, or be devoid of that flanking sequence with few mismatches at the end toward the flanking side (as generated with PCR using 59 and 39 extreme genespecific primers containing a different restriction site toward the flanking side or as generated with any of the convenient methods to avoid extension toward the flanking side) (10 –12). In the present study template A contains the TP2 coding sequence in the background of pET-22b from which the vector-specific primer is used as the 59 end primer (S1) containing the appropriate restriction site (XbaI) for subsequent subcloning of the PCR product and template B contains the TP2 coding sequence in the background of pTrc 99A. Primer S2 is an insert-specific primer containing an appropriate restriction site (HindIII) which binds at the 39 extreme end of TP2 cDNA. All the mutagenic primers, m1 to
FIG. 2. Ethidium bromide-stained agarose gel pattern of PCR products generated after each step of mutagenesis. M, molecular size marker. Lanes 1–7, mutant products after the first and subsequent PCR.
290
NOTES & TIPS
m6, were designed by taking care of all the parameters for proper annealing to the wild-type template. All the mutagenic primers are in the same orientation such that the PCR product will be formed with each mutagenic primer and only one common side primer (S1). PCR is initiated with the mutagenic primer which is nearest to the side primer S1 and as the size of the PCR product increases with each of the new mutagenic primers (extension of megaprimer method, Refs. 7–9), the resulting new PCR product will have two, three, . . . , etc. site-specific mutations. First PCR. The PCR mixture contained 2 ng of template A, 8 pmol each of primers S1 and m1, 10 nmol of dNTPs and 1 unit of Pfu polymerase (Stratagene) in a total reaction volume of 50 ml (Perkin–Elmer Gene Amp PCR System 2400). The cycling conditions were 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min for a total of 25 cycles followed by a further extension at 72°C for 3 min. Second PCR. Template A was removed from the first PCR product by gel elution from 5% PAGE. This step is very crucial to achieve high efficiency of the method. Without this step wild-type template will also be amplified in the subsequent PCR. This is the only step where gel purification is required in the entire procedure. The PCR contained 5 ng of purified PCR product P1 and 2 ng of template B, 8 pmol each of primer S1 and second mutagenic primer m2, 10 nmol of dNTPs, and 1 unit of Pfu polymerase and the cycling conditions were the same as described above. Third and subsequent PCR. For the third PCR, the first 5 cycles of PCR were carried out with 5 ng of unpurified PCR product P2, 2 ng of template B, and other components except primers S1 and m3. This step allows the extension of the 39 end of P2 along the template, thereby enriching the template having two mutations before adding the primers. In this step few template molecules which were extended from P1 (having only one mutation) will be outnumbered by the template containing two mutations. After 5 cycles of PCR, 8 pmol each of primers S1 and m3 was added and the reaction was allowed to proceed for another 25 cycles. These steps were repeated with subsequent mutagenic primers. In the last step the full-length PCR product was obtained by using primers S1 and S2 and subcloned into the desired vector (XbaI and HindIII sites of pET-22b, in this study). DNA from few of the clones obtained were sequenced by dideoxy-terminator chemistry in a Perkin–Elmer 377A automated DNA sequencer. Figure 1 illustrates the overall strategy designed to create multiple site-specific mutations in the TP2 gene. Figure 2 shows the ethidium bromide-stained agarose gel pattern of PCR products obtained at each step of the PCR. It is very clear that each of the mutagenic
primers has yielded the next sized product with very little of the previous PCR product left behind. After incorporation of six site-specific mutations through six rounds of PCR, we sequenced the full-length PCR product and observed more than 80% incorporation of all six mutations (data not shown). Subsequently, we sequenced 10 clones after subcloning the PCR product into pET-22b and observed that 8 clones have all six mutations while 2 clones had five mutations (data not shown). Both clones missed one mutation each at the same site. This might be due to the inefficient annealing of the mutant primer m4 under the same PCR condition. Thus, the frequency of clones containing all of the desired mutations may vary with the efficiency of priming by the mutagenic primer in each round of PCR. However, since each PCR is independent of others, the annealing condition can be optimized with respect to each specific mutagenic primer to improve further the efficiency of the present method. It is also possible to generate any combination of multiple mutations by selecting the appropriate mutagenic primers in the same way. Since we used Pfu polymerase for our PCR and the total size of the gene is small, we could not detect any secondary mutations in the entire TP2 cDNA. In summary, this communication describes a simple and efficient method of creating multiple site-specific mutations in a given gene without going through the necessity of cloning the PCR product at each step of PCR mutagenesis. Acknowledgments. This work was financially supported by Department of Atomic Energy, Government of India. A. R. Meetei is a Senior Research Fellow of CSIR. DNA sequencing was carried out in the DBT-supported automated DNA sequencing facility.
REFERENCES 1. Ling, M. M., and Robinson, B. H. (1997) Anal. Biochem. 254, 157–178. 2. Higuchi, R., Krummel, B. K., and Saiki, R. K. (1988) Nucleic Acids Res. 16, 7351–7367. 3. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 51–59. 4. Landt., O., Grunert, H. P., and Hahn, U. (1990) Gene 96, 125– 128. 5. Sankar, G., and Sommer, S. S. (1990) BioTechniques 8, 404 – 407. 6. Marini, F., III, Naeem, A., and Lapeyre, J. N. (1993) Nucleic Acids Res. 21, 2277–2278. 7. Good, L., and Nazar, R. N. (1992) Nucleic Acids Res. 20, 4934. 8. Steinberg, R. A., and Gorman, K. B. (1994) Anal. Biochem. 219, 155–157. 9. Datta, A. K. (1995) Nucleic Acids Res. 23, 4530 – 4531. 10. Barettino, D., Feigenbutz, M., Valcarcel, R., and Stunnenberg, H. G. (1994) Nucleic Acids Res. 22, 541–542. 11. Upender, M., Raj, L., and Weir, M. (1995) BioTechniques 18, 29 –30. 12. Boles, E., and Miosga, T. (1995) Curr. Genet. 28, 197–198.
NOTES & TIPS
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13. Seraphin, B., and Kandels-Lewis, S. (1996) Nucleic Acids Res. 24, 3276 –3277. 14. Baskaran, R., and Rao, M. R. S. (1991) Biochem. Biophys. Res. Commun. 179, 1491–1499. 15. Kundu, T. K., and Rao, M. R. S. (1995) Biochemistry 34, 5143– 5150. 16. Kundu, T. K., and Rao, M. R. S. (1996) Biochemistry 35, 15626 – 15632. 17. Meetei, A. R., and Rao, M. R. S. (1996) Protein Express. Purif. 8, 409 – 415. 18. Meetei, A. R., and Rao, M. R. S. (1998) Protein Express. Purif. 13, 184 –190. 19. Dwivedi, U. N., Shiraishi, N., and Campbell, W. H. (1994) Anal. Biochem. 221, 425– 428. 20. Tu, H. M., and Sun, S. S. M. (1996) BioTechniques 20, 352–353. 21. Bi, W., and Stambrook, P. J. (1998) Anal. Biochem. 256, 137– 140.
A Combined Flow Injection±Chemiluminescent Method for the Measurement of Radical Scavenging Activity
FIG. 1. The effect of H2O2 on CL responses. The amount of 0.06% H2O2 injected: (A) Blank, (B) 2 ml, (C) 3 ml, (D) 4 ml, (E) 5 ml. The average peak height of 5 ml was arbitrarily set at 1000.
Hong-Yeob Choi, Jin-Hyang Song, and Dong-Ki Park1 Department of Biochemistry, Kon-Kuk University, Chungju, Chungbuk 380-701, Korea
We report here a simple, rapid and reproducible flow injection–CL (FI–CL) method for the measurement of radical scavenging ability.
Received June 9, 1998
Materials and Methods Oxygen-derived free radicals, such as the hydroxyl radical (OH•), singlet oxygen (1O2), and superoxide anion (O2. ), have been known as potent agents causing various pathological effects including aging, atherosclerosis, and even cancer (1, 2). Therefore, many kinds of natural or synthetic radical scavengers have been reported and used to protect biological molecules from the radicals. Also, a number of methods have been developed for the determination of radical scavenging activity: electron spin resonance spectroscopy (3), a pulse radiolysis technique (4), the measurement of redox potentials (5), and a chemiluminescent method. Among them, chemiluminescence (CL)2 offers great advantages over other techniques; it is extremely sensitive, simple, and rapid. Hirayama and Yida (6) developed a method for determining OH• generated by Fenton reaction and applied this method to evaluate the radical scavenging activity of biological components. To whom correspondence should be addressed. Fax: 182-441851-0242. E-mail: [email protected]. 2 Abbreviations used: CL, chemiluminescence; FI, flow injection; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; SOD, superoxide dismutase; RSD, relative standard deviation. 1
ANALYTICAL BIOCHEMISTRY ARTICLE NO. AB982864
264, 291–293 (1998)
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Reagents. Cytochrome c, luminol, gallic acid, and hypoxanthin were purchased from Sigma Chemical Co. (St. Louis, MO). Hydrogen peroxide was obtained from Hayashi Pure Chemical Industry (Osaka, Japan). 5,5Dimethyl-1-pyrroline-N-oxide (DMPO) was obtained from Labotec Co. (Tokyo, Japan). Xanthin oxidase and superoxide dismutase (SOD) were purchased from Boehringer Mannhein Co. (Tokyo, Japan). Measurement of chemiluminescence by the FI–CL system. The CL was measured with the filterequipped photon counting type spectrophotometer (CLD-110, Tohoku Electronic Industry) connected to a pump (Gilson Model 303) and a sample injection valve (Rheodyne Model 7125). The dispersed light at the grating was simultaneously detected on the photocathode with the image sensor in the range of 300 – 650 nm. The photons counted in the range of 300 – 650 nm were computed as a total spectral intensity. The mobile phase was 50 mM phosphate buffer (pH 7.4) containing 50% methanol (for solvent-soluble samples), cytochrome c (10 mg/liter), and luminol (2 mg/liter); the flow rate was maintained at 1.0 ml/min with the pump. For the purpose of measuring the abilities of radical scavengers, the mixture of 0.06% H2O2 (5 ml) and scav-
NOTES & TIPS
under the same assay conditions) the activities at 30 and 40°C. Assay with the eluted protein at 50°C showed the presence of 75.96 units of glucoamylase and 0.72 units a-D-glucosidase bound to the immobilized antibody. Since the bound enzyme could be eluted from the antibody at 50°C and the eluted enzymes were pure as seen by SDS–PAGE after silver staining of the gels (data not shown), the procedure could be applied for activity determinations at higher temperatures. These experiments showed a temperature optima at 60°C for glucoamylase and a-D-glucosidase (Fig. 2) defining applicability of the procedure for thermostability determinations of enzymes. The procedure avoided harsh treatments generally used for elution procedures (11). The elution is apparently due to temperature and can be employed with enzymes stable at higher temperatures. The temperature optima described of these enzymes were significantly different from the assay carried out with crude culture filtrates (Fig. 2). The interfering starch hydrolyzing enzymes contributed to increased temperature activities because we could purify an a-amylase active at acidic pH from the culture fluids and a-D-glucosidase showed activities on starch and dextrins but with different substrate specificities as defined by Km (10). Though the procedure involves an antibody, for enzyme-engineering studies, a single antibody preparation can be used to selectively isolate clones with improved activities at high temperature without interferences by other substances in the crude broth. Acknowledgments. The authors thank the director of the institute for facilities. This research was supported by the Council of Scientific and Industrial Research, New Delhi, through Junior Research Fellowships to C.S., A.K.D., and R.K.
REFERENCES 1. Frandsen, P. T., Christensen, T., Stoffer, B., Lehmbeck, J., Dupont, C., Honzatko, R. B., and Svensson, B. (1995) Biochemistry 34, 10162–10169. 2. Christensen, U., Olsen, K., Stofer, B. B., and Svennson, B. (1996) Biochemistry 35, 15009 –15018. 3. Spector, T. (1978) Anal. Biochem. 86, 142–146. 4. Shankar, C. S., and Umesh-Kumar, S. (1994) Microbiology 140, 1097–1101. 5. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. 6. Laemmli, U. K. (1970) Nature 227, 680 – 685. 7. Batteiger, B., Newhall, W. J., and Jones, R. B. (1982) J. Immunol. Methods 55, 297–307. 8. Dahlqvist, A. (1961) Biochem. J. 80, 547–551. 9. Boel, E., Hjort, I., Svensson, B., Norris, F., Norris, K. E., and Fill, N. P. (1984) EMBO J. 3, 1097–1102. 10. Kita, A., Matsui, H., Samoto, A., Kimura, A., Takata, M., and Chiba, S. (1991) Agric. Biol. Chem. 55, 2327–2335. 11. Thomas, C. T., and McNamee, M. G. (1990) Methods Enzymol. 182, 499 –515.
Generation of Multiple Site-Specific Mutations in a Single Polymerase Chain Reaction Product Amom Ruhikanta Meetei and M. R. S. Rao Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Received May 13, 1998
Site-directed mutagenesis has been very valuable in studying the structure–function relationship of proteins (1). PCR-mediated mutagenesis has been the method of choice in recent years to change the desired amino acid from one to another. Several procedures have been described to achieve the desired mutation using PCR technology (2–13). Although in most of the cases the desired change is only with respect to one amino acid, sometimes one needs to generate several changes in the amino acid sequence to understand the biological function of a given protein. Such a necessity is apparent when one is dealing with a zinc finger class of proteins wherein multiple residues of cysteine and histidine are involved in the coordination of zinc. We have been working over the past several years on the structure–function relationship of a spermatidal specific protein TP2 which appears transiently during mammalian spermiogenesis. We have shown that this basic nuclear protein binds two atoms of zinc involving both cysteine and histidine residues (14) and this zincinduced polypeptide fold is necessary for interaction and condensation of DNA with a preference to alternating GC containing DNA (15). Furthermore, this observation led to our study demonstrating that TP2, in the zinc coordinated form, recognizes a human CpG island sequence (16). The amino acid sequences of rat, mouse, and human TP2 contain several cysteine and histidine residues in the N-terminal two-thirds of the molecule, more than what is required for coordination of two zinc atoms. Amino acid sequence alignment of the zinc binding domain of TP2 revealed that it does not have strong similarity with any of the known classes of zinc finger proteins. This has posed a challenge to identify the actual residues involved in zinc coordination. We have recently cloned and expressed rat TP2 in Escherichia coli which has paved the way for answering this question (17, 18). During this course of investigation, we had to develop multiple mutants in the same molecule. There are at least three reports describing methods for generating the multiple sitespecific mutations (19 –21). The first method described by Dwivedi et al. (19) is termed as the MM-SSP method in which double mutations were created by using the previous single-mutant clone as starting template and so forth. This method involves confirmation of each ANALYTICAL BIOCHEMISTRY 264, 288 –291 (1998) ARTICLE NO. AB982866
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS
FIG. 1. A schematic representation of the strategy for generating multiple site-specific mutations. S1, side primer; P1, P2, . . . , P6, mutagenic PCR products; m1, m2, . . . , m6, mutagenic primers; and S2, 39 end specific primer.
mutant clone every time before starting the next mutation. In the second method described by Tu and Sun (20), different mutagenic primers were used at a time to create a combination of mutations in a given gene. However, this method also involved generation of single-stranded DNA template containing dUMP and subsequent screening of mutant clones (essentially an extension of Kunkel’s method). Furthermore, the efficiency of mutagenesis was not very high. Recently, CCR (combined chain reaction) was developed by Bi and Stambrook (21) for generating single and multiple mutations. In this method, one needs special considerations, such as annealing efficiency of mutant primers should not be less than that of side primers and DNA polymerase should not have 59 to 39 exonuclease activity. Although this method appears highly efficient to generate single and double mutations, the efficiency of generation of three or more multiple mutations needs to be tested. In all three methods there is an absolute
289
requirement of DNA ligase (thermostable ligase in the third method) and 59 phosphorylation of mutagenic primers. We report here a simple and very efficient method to generate multiple site-specific mutations in a gene of interest wherein no DNA ligase, no 59 phosphorylation of mutagenic primers, no consideration of annealing efficiency of mutagenic primers compared with side primers in the same PCR condition, no extra care for 59 to 39 exonuclease activity of thermostable DNA polymerase, and no subcloning of PCR product each time the mutagenic primer is used are required. This method has also allowed us to create only the desired multiple mutations and not a combination of multiple mutations. One of the important requirements of the present method is to have two templates (A and B) in which one of the templates has an extra flanking sequence in addition to the gene or part of the gene of interest, to which one of the side primers (S1) binds while the other template has no binding site for this side primer. The second template can either have a different flanking side, as generated from two different vector backgrounds, or be devoid of that flanking sequence with few mismatches at the end toward the flanking side (as generated with PCR using 59 and 39 extreme genespecific primers containing a different restriction site toward the flanking side or as generated with any of the convenient methods to avoid extension toward the flanking side) (10 –12). In the present study template A contains the TP2 coding sequence in the background of pET-22b from which the vector-specific primer is used as the 59 end primer (S1) containing the appropriate restriction site (XbaI) for subsequent subcloning of the PCR product and template B contains the TP2 coding sequence in the background of pTrc 99A. Primer S2 is an insert-specific primer containing an appropriate restriction site (HindIII) which binds at the 39 extreme end of TP2 cDNA. All the mutagenic primers, m1 to
FIG. 2. Ethidium bromide-stained agarose gel pattern of PCR products generated after each step of mutagenesis. M, molecular size marker. Lanes 1–7, mutant products after the first and subsequent PCR.
290
NOTES & TIPS
m6, were designed by taking care of all the parameters for proper annealing to the wild-type template. All the mutagenic primers are in the same orientation such that the PCR product will be formed with each mutagenic primer and only one common side primer (S1). PCR is initiated with the mutagenic primer which is nearest to the side primer S1 and as the size of the PCR product increases with each of the new mutagenic primers (extension of megaprimer method, Refs. 7–9), the resulting new PCR product will have two, three, . . . , etc. site-specific mutations. First PCR. The PCR mixture contained 2 ng of template A, 8 pmol each of primers S1 and m1, 10 nmol of dNTPs and 1 unit of Pfu polymerase (Stratagene) in a total reaction volume of 50 ml (Perkin–Elmer Gene Amp PCR System 2400). The cycling conditions were 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min for a total of 25 cycles followed by a further extension at 72°C for 3 min. Second PCR. Template A was removed from the first PCR product by gel elution from 5% PAGE. This step is very crucial to achieve high efficiency of the method. Without this step wild-type template will also be amplified in the subsequent PCR. This is the only step where gel purification is required in the entire procedure. The PCR contained 5 ng of purified PCR product P1 and 2 ng of template B, 8 pmol each of primer S1 and second mutagenic primer m2, 10 nmol of dNTPs, and 1 unit of Pfu polymerase and the cycling conditions were the same as described above. Third and subsequent PCR. For the third PCR, the first 5 cycles of PCR were carried out with 5 ng of unpurified PCR product P2, 2 ng of template B, and other components except primers S1 and m3. This step allows the extension of the 39 end of P2 along the template, thereby enriching the template having two mutations before adding the primers. In this step few template molecules which were extended from P1 (having only one mutation) will be outnumbered by the template containing two mutations. After 5 cycles of PCR, 8 pmol each of primers S1 and m3 was added and the reaction was allowed to proceed for another 25 cycles. These steps were repeated with subsequent mutagenic primers. In the last step the full-length PCR product was obtained by using primers S1 and S2 and subcloned into the desired vector (XbaI and HindIII sites of pET-22b, in this study). DNA from few of the clones obtained were sequenced by dideoxy-terminator chemistry in a Perkin–Elmer 377A automated DNA sequencer. Figure 1 illustrates the overall strategy designed to create multiple site-specific mutations in the TP2 gene. Figure 2 shows the ethidium bromide-stained agarose gel pattern of PCR products obtained at each step of the PCR. It is very clear that each of the mutagenic
primers has yielded the next sized product with very little of the previous PCR product left behind. After incorporation of six site-specific mutations through six rounds of PCR, we sequenced the full-length PCR product and observed more than 80% incorporation of all six mutations (data not shown). Subsequently, we sequenced 10 clones after subcloning the PCR product into pET-22b and observed that 8 clones have all six mutations while 2 clones had five mutations (data not shown). Both clones missed one mutation each at the same site. This might be due to the inefficient annealing of the mutant primer m4 under the same PCR condition. Thus, the frequency of clones containing all of the desired mutations may vary with the efficiency of priming by the mutagenic primer in each round of PCR. However, since each PCR is independent of others, the annealing condition can be optimized with respect to each specific mutagenic primer to improve further the efficiency of the present method. It is also possible to generate any combination of multiple mutations by selecting the appropriate mutagenic primers in the same way. Since we used Pfu polymerase for our PCR and the total size of the gene is small, we could not detect any secondary mutations in the entire TP2 cDNA. In summary, this communication describes a simple and efficient method of creating multiple site-specific mutations in a given gene without going through the necessity of cloning the PCR product at each step of PCR mutagenesis. Acknowledgments. This work was financially supported by Department of Atomic Energy, Government of India. A. R. Meetei is a Senior Research Fellow of CSIR. DNA sequencing was carried out in the DBT-supported automated DNA sequencing facility.
REFERENCES 1. Ling, M. M., and Robinson, B. H. (1997) Anal. Biochem. 254, 157–178. 2. Higuchi, R., Krummel, B. K., and Saiki, R. K. (1988) Nucleic Acids Res. 16, 7351–7367. 3. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 51–59. 4. Landt., O., Grunert, H. P., and Hahn, U. (1990) Gene 96, 125– 128. 5. Sankar, G., and Sommer, S. S. (1990) BioTechniques 8, 404 – 407. 6. Marini, F., III, Naeem, A., and Lapeyre, J. N. (1993) Nucleic Acids Res. 21, 2277–2278. 7. Good, L., and Nazar, R. N. (1992) Nucleic Acids Res. 20, 4934. 8. Steinberg, R. A., and Gorman, K. B. (1994) Anal. Biochem. 219, 155–157. 9. Datta, A. K. (1995) Nucleic Acids Res. 23, 4530 – 4531. 10. Barettino, D., Feigenbutz, M., Valcarcel, R., and Stunnenberg, H. G. (1994) Nucleic Acids Res. 22, 541–542. 11. Upender, M., Raj, L., and Weir, M. (1995) BioTechniques 18, 29 –30. 12. Boles, E., and Miosga, T. (1995) Curr. Genet. 28, 197–198.
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A Combined Flow Injection±Chemiluminescent Method for the Measurement of Radical Scavenging Activity
FIG. 1. The effect of H2O2 on CL responses. The amount of 0.06% H2O2 injected: (A) Blank, (B) 2 ml, (C) 3 ml, (D) 4 ml, (E) 5 ml. The average peak height of 5 ml was arbitrarily set at 1000.
Hong-Yeob Choi, Jin-Hyang Song, and Dong-Ki Park1 Department of Biochemistry, Kon-Kuk University, Chungju, Chungbuk 380-701, Korea
We report here a simple, rapid and reproducible flow injection–CL (FI–CL) method for the measurement of radical scavenging ability.
Received June 9, 1998
Materials and Methods Oxygen-derived free radicals, such as the hydroxyl radical (OH•), singlet oxygen (1O2), and superoxide anion (O2. ), have been known as potent agents causing various pathological effects including aging, atherosclerosis, and even cancer (1, 2). Therefore, many kinds of natural or synthetic radical scavengers have been reported and used to protect biological molecules from the radicals. Also, a number of methods have been developed for the determination of radical scavenging activity: electron spin resonance spectroscopy (3), a pulse radiolysis technique (4), the measurement of redox potentials (5), and a chemiluminescent method. Among them, chemiluminescence (CL)2 offers great advantages over other techniques; it is extremely sensitive, simple, and rapid. Hirayama and Yida (6) developed a method for determining OH• generated by Fenton reaction and applied this method to evaluate the radical scavenging activity of biological components. To whom correspondence should be addressed. Fax: 182-441851-0242. E-mail: [email protected]. 2 Abbreviations used: CL, chemiluminescence; FI, flow injection; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; SOD, superoxide dismutase; RSD, relative standard deviation. 1
ANALYTICAL BIOCHEMISTRY ARTICLE NO. AB982864
264, 291–293 (1998)
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
Reagents. Cytochrome c, luminol, gallic acid, and hypoxanthin were purchased from Sigma Chemical Co. (St. Louis, MO). Hydrogen peroxide was obtained from Hayashi Pure Chemical Industry (Osaka, Japan). 5,5Dimethyl-1-pyrroline-N-oxide (DMPO) was obtained from Labotec Co. (Tokyo, Japan). Xanthin oxidase and superoxide dismutase (SOD) were purchased from Boehringer Mannhein Co. (Tokyo, Japan). Measurement of chemiluminescence by the FI–CL system. The CL was measured with the filterequipped photon counting type spectrophotometer (CLD-110, Tohoku Electronic Industry) connected to a pump (Gilson Model 303) and a sample injection valve (Rheodyne Model 7125). The dispersed light at the grating was simultaneously detected on the photocathode with the image sensor in the range of 300 – 650 nm. The photons counted in the range of 300 – 650 nm were computed as a total spectral intensity. The mobile phase was 50 mM phosphate buffer (pH 7.4) containing 50% methanol (for solvent-soluble samples), cytochrome c (10 mg/liter), and luminol (2 mg/liter); the flow rate was maintained at 1.0 ml/min with the pump. For the purpose of measuring the abilities of radical scavengers, the mixture of 0.06% H2O2 (5 ml) and scav-