Benchmarks monas aeruginosa using frozen cell suspensions. FEMS Microbiol. Lett. 58:221-225. 5.Fiedler, S. and R. Wirth. 1988. Transformation of bacteria with plasmid DNA by electroporation. Anal. Biochem. 170:38-44. 6.Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580. 7.Holloway, B.W., V. Krishnapillai and A.F. Morgan. 1979. Chromosomal genetics of Pseudomonas. Microbiol. Rev. 43:73-102. 8.Itoh, N., T. Kouzai and Y. Koide. 1994. Efficient transformation of Pseudomonas strains with pNI vectors by electroporation. Biosci. Biotechnol. Biochem. 58:1306-1308. 9.Kilbane, J.J., II. and B.A. Bielaga. 1991. Instantaneous gene transfer from donor to recipient microorganisms via electroporation. BioTechniques 10:354-365. 10.Pederson, N.E. 1996. Spin-column chromatography for DNA purification. Anal. Biochem. 239:117-118. 11.Smith, A.W. and B.H. Iglewski. 1989. Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res. 17:10509. 12.West, S.E., H.P. Schweizer, C. Dall, A.K. Sample and L.J. Runyen-Janecky. 1994. Construction of improved EscherichiaPseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81-86. 13.Yanisch-Perron, C., J. Vieira and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119.
PCR-Based Random Mutagenesis Method Using Spiked Oligonucleotides to Randomize Selected Parts of a Gene without any Wild-Type Background BioTechniques 25:958-965 (December 1998)
Site-directed mutagenesis (SDM) is a common tool to analyze protein structure and function (2). In polymerase chain reaction (PCR)-based SDM methods, the mutation is introduced by a PCR primer (1,3–7). Also, a restrictionenzyme-marker site is often introduced to allow fast and convenient screening for the presence of the mutation (4,7,9). Usually, each primer encodes only one particular amino acid (aa) exchange. In principle then, it is sufficient to identify
one marker-positive clone. Under these circumstances, the level of background of nonmutated genes is not important considering that more than approximately 20% of the clones carry the mutation. If only 20% of the transformants contained a mutated gene, then screening 10–20 of the clones would be sufficient to identify at least one mutant, with a probability of 90%–99%. However, the approach of introducing nucleotide exchanges into a gene by a PCR primer can be easily extended to randomize larger parts of the gene if spiked oligonucleotides, which contain some degree of a mixture of all 4 nucleotides, are used (8,10). Then, one usually intends to isolate as many mutant clones as possible to obtain as large a library of mutated genes as possible. Because it is impossible with respect to time and cost to screen more than a few hundred clones, a method for mutagenesis that excludes the wild-type (WT) genes from the transformants with high confidence is required.
The authors thank Drs. Paul Hager and Paul Phibbs, East Carolina University School of Medicine, for the gifts of the Pseudomonas strain and plasmid. Address correspondence to Dr. Mary A. Farwell, Department of Biology, East Carolina University, Greenville, NC 27858, USA. Internet:
[email protected] Received 3 April 1998; accepted 10 August 1998.
Patrick J. Enderle and Mary A. Farwell East Carolina University Greenville, NC, USA
Figure 1. Schematic drawing of the annealing positions of the primers used for PCR mutagenesis of the codons 95–104 of the EcoRV gene. In addition, the PCR products obtained in the different PCRs are shown. For mutagenesis of codons 180–184, an NgoMI site was used as a marker/cloning site.
Benchmarks Here, we describe a random mutagenesis protocol that allows the construction of large mutant libraries (104–105 clones) without any WT background and therefore without the necessity to screen for the presence of mutations. This method comprises two steps. In step 1, PCR mutagenesis was carried out using a well-established megaprimer technique (4,9). The mutagenesis protocol used a linear template carrying the target gene flanked by an NsiI site upstream and a SalI site downstream, three common primers: Pup, Plow1 (removing the SalI site) and Plow2 (preserving the SalI site), and an additional set of primers, one for each region of the gene subjected to mutagenesis (Pmut). These mutagenic primers contain a small amount of all 4 nucleotides at each position to be mutated and also introduce a restriction-enzyme-marker site (ClaI or NgoMI) upstream to the randomized region (Figure 1). In a PCR with Pup and Plow1 (PCR I), a full-length PCR product of the EcoRV gene without the SalI site is obtained (Figure 1, product I). Using Pmut and Plow2, product II was amplified in PCR II. It comprises the 3′ part of the gene, including the region to be mutated (Figure 1). The SalI site is preserved in this fragment, and the marker site is introduced by Pmut upstream of the region containing the mutations. Then, both PCR products (I and II) were hybridized, producing heteroduplexes. One heteroduplex, consisting of the upper strand of product I and the lower strand of product II, can be extended at the 3′ ends of both strands by the Pfu DNA Polymerase (Stratagene, La Jolla, CA, USA) obtaining complete EcoRV genes. Then the EcoRV genes were amplified with Pup and Plow2 (PCR III), and all products containing a SalI site at the 3′ end of the gene should carry a mutation (Figure 1, product III). The PCR products were digested with SalI and NsiI and ligated into the large fragment of pHis, which was digested with the same enzymes. The ligation product was used to transform E. coli cells (9). This is a very robust, fast, cost-effective and reliable method, but differs in practice from theoretical expectations because it does not lead to a yield of 100% mutated clones. In the experiments, usually only 20%–40% of
Figure 2. Generation of a library of EcoRV variants without WT background. Starting with the vector pHis carrying the EcoRV WT gene, a library of mutated genes that does not contain any WT genes is prepared in a two-step procedure. Note that product III and product II are randomized at one target region.
Benchmarks Table 1. Experimental Procedures
All PCRs were performed using 200 µM dNTPs, 400 nM of each primer and 2 U native Pfu DNA Polymerase in a total volume of 50 µL (Pfu DNA polymerase was used to minimize mutations outside the target regions). PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Step 1 In PCR I, 50 ng linearized pHis carrying the EcoRV gene were used as a template and amplified with: Pup (5′-GGAGAAATTAACTGCATGCATCATC-3′) and Plow1 (5′-GACGTCCAGCGGCCTAGGCCACGAT-3′). PCR conditions were 2 min at 95°C followed by 15 cycles of 30 s at 95°C, 1 min at 52°C and 1 min at 72°C. Approximately 200 ng product I were obtained. An analogous PCR (PCR II) (2 min at 95°C followed by 15 cycles of 30 s at 95°C, 1 min at 55°C and 1 min at 72°C) was carried out using: Pmut1 (5′-AAAAATTGCAATCGATATAAAAACAACATATACAAACAAAGAAAACGAAAAAATCAAGTTCACTCTTGG-3′—the ClaI site is given in bold face, and the region underlined is degenerated by 6%) or Pmut2 (5′-ATGGGTTATTGCCGGCGATTTGGCAGGATCTGGAAACACAACAA-3′—the NgoMI site is given in bold face, and the region underlined is degenerated by 14%) and Plow2 (5′-AGTTGTCCTCAGGTTCGAGTCGATT-3′). Typically 200 ng product II were obtained. 10–50 ng of product I and product II were used in the third PCR using primers Pup and Plow2 (3 cycles of 1 min at 95°C, 1 min at 60°C and 2 min at 72°C; 15 cycles of 30 s at 95°C, 1 min at 54°C and 1 min at 72°C). During the first 3 cycles of denaturation and reannealing (under these conditions primer binding is disfavored), the heteroduplexes were filled up by the Pfu DNA polymerase. The resulting products were amplified in the following cycles. Typical yields of this reaction were between 500 ng and 1 µg DNA. The purified full-length products were digested with 25 U of SalI (MBI Fermentas, Vilnius, Lithuania) and NsiI (MBI Fermentas) for 2 h in 50 µL at 37°C, purified with QIAquick to remove the restriction enzymes and the short cleavage fragments, ligated into the large SalI/NsiI fragment of pHis using 1 U T4 Ligase (MBI Fermentas) at 16°C for 16 h and then transformed into E. coli cells. Plasmids from several clones were prepared and analyzed with ClaI or NgoMI for the presence of the marker site. Step 2 30 µg plasmid of one marker positive clone were digested with ClaI or NgoMI and SalI. Cleavage products were then separated on an agarose gel, and the large fragment was purified from the gel using Silica Beads (No. 101270; Merck, Darmstadt, Germany) according to the instructions of the supplier. PCR product II was digested with ClaI or NgoMI and SalI and then ligated into the large ClaI/SalI or NgoMI/SalI fragment of pHis using 1 U T4 Ligase at 16°C for 16 h and transformed into E. coli cells. All transformants contained the desired mutations. Both strands of the complete EcoRV gene of 28 clones were sequenced using a Model 373 DNA Sequencer (PE Applied Biosystems, Foster City, CA, USA).
Table 2. Results of the Screening of EcoRV Variants Obtained in Different Rounds of Random Mutagenesis
WT Background of Screened Clones Length of PCR product II (marker site)
Before second stepa
After second stepb
650 (ClaI)
56 of 70
0 of 16
386 (NgoMI)
31 of 45
0 of 12
aScreened bScreened
for presence of the marker site. for presence of the marker site and sequenced.
the obtained clones contained the marker site and thus, the desired mutations (Table 2; cf. References 3 and 9). This WT background might be explained by different models. Some of the WT EcoRV genes used in the first 2 PCRs as a template might not have been removed during the purification of product I and II. These molecules could then have acted as templates in the last PCR with Pup and Plow2 and lead to the WT background. Alternatively, template switching of the Pfu DNA polymerase during PCR III could
produce EcoRV genes that contain a SalI site but no mutations. To construct a large library of mutated genes without the WT background, we have developed a second step (step 2) as an addition to the existing protocol. In this step, the marker site is used as the cloning site (Figure 2). After step 1, only one marker-positive clone must be isolated, and the plasmid of this clone digested with the marker enzyme and SalI to generate a new large fragment that still carries the part of the EcoRV gene upstream of the marker site. The PCR product obtained in PCR II of step 1 was cleaved with the marker enzyme and SalI and ligated into the large fragment. The ligation product was then used to transform E. coli cells. This procedure leads to a zero WT background in the obtained clones, because only PCR products containing the desired mutations can carry a marker site. Any PCR products amplified from contaminating WT genes, irrespective to the origin of the contamination, cannot be cloned. We mutated the gene of the restriction endonuclease EcoRV using this procedure in two regions, with each region having its own mutagenic primer and restriction enzyme marker site (ClaI or NgoMI). Plasmid preparations of the obtained libraries were completely digested by the marker enzyme in both cases. In all individual clones isolated from both libraries, no single WT gene was detected (Table 2). The only possible unwanted outcome of this procedure could be an overrepresentation of the clones used to prepare the large ClaI/SalI or NgoMI/SalI fragments. However, in 28 sequenced clones, the original clone was never observed a second time, demonstrating that the experimental procedures used here to purify the fragments from an agarose gel can reliably prevent this kind of contamination. Note that step 2 is an optional addition to the well-established protocol for the site-directed PCR mutagenesis carried out in step 1. In practice, step 2 needs only to be carried out if the WT background obtained after step 1 is too high for experimental purposes. Instead of repeating step 1, and possibly repeating the same mistakes, our protocol provides an easy tool to eliminate any WT background.
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Address correspondence to Dr. Albert Jeltsch, Institut für Biochemie, FB Biologie, Justus-Liebig Universität, Heinrich-BuffRing 58, 35392 Giessen, Germany. Internet:
[email protected] Received 11 May 1998; accepted 21 August 1998.
Thomas Lanio and Albert Jeltsch Justus-Liebig Universität Giessen, Germany
