Abstract
Three mutants of the wild type α-amylase gene from Xanthomonas campestris pv. campestris 8004 were obtained using a PCR technique in which deoxythymidine triphosphate (dTTP) was partially replaced by 5-bromo-2′-deoxyuridine-5′-triphosphate (BrdUTP), at an optimal dTTP:BrdUTP ratio of 1000:1. Of thre three mutants that were obtained and which were sequenced, one mutant with 40 times higher activity than the wild type α-amylase gene product was obtained by using primary PCR products as a template for a second PCR reaction.
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References
Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359.
Hu NT, Hung MN, Hung AM (1992) Molecular cloning, characterization and nucleotide sequence of the gene for secreted á-amylase from Xanthomonas campestris pv. Campestris. J. Gen. Microbiol. 138: 1647–1655.
Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96: 23–28.
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82: 488–492.
Leung DW, Chen E, Goeddel DV (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Techniques 1: 11–15.
Ma XD, Ma LX, Xue ZF, Zhou JC (2000) A new method to identify á-amylase activity and its producing bacteria. J. Huazhong Agric. Univ. 19: 456–460 (in Chinese with English summary).
Sambrook J, Fritsch EF, Maniatias T (1992) Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory.
Sigal L, Harwood BG, Arentzen R, Betty GH, Rene A (1982) Thiol-â-lactamase replacement of the active-site serine of RTEM â-lactamase by a cysteine residue. Proc. Natl. Acad. Sci. USA 79: 7157–7160.
Solnick D (1981) An adenovirus mutant defective in splicing RNA from early region IA. Nature 291: 508–510.
Stemmer WPC (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370: 389–391.
Weiner MP, Costa GL, Schoettlin W, Cline J, Mathur E, Bauer JC (1994) Site-directed mutagenesis of double-stranded DNA by the polymetase chain reaction. Gene 151: 119–123.
Wells TA, Vasser M, Powers DB (1985) Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites.Gene 34: 315–323.
Zhang JH, Dawes G, Stemmer WPC (1997) Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl. Acad. Sci. USA 94: 4504–4509.
Zhao H, Giver L, Shao Z, Affholter JA, Arnold FH (1998) Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16: 258–261.
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Ma, X., Ke, T., Li, Y. et al. In vitro mutagenesis of Xanthomonas campestris α-amylase gene by partially replacing deoxythymidine triphosphate with 5-bromo-2′-deoxyuridine-5′-triphosphate using a PCR technique. Biotechnology Letters 26, 171–175 (2004). https://doi.org/10.1023/B:BILE.0000012901.89522.20
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DOI: https://doi.org/10.1023/B:BILE.0000012901.89522.20