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KMID : 1007520030120010104
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Food Science and Biotechnology
2003 Volume.12 No. 1 p.104 ~ p.106
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Megaprimer PCR for Site-directed Mutagenesis of Cyclodextrin Glucanotransferase
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Kim, Chang Sup
Han, Nam Soo/Keum, Inkyung/Tao, Bernard Y./Seo, Jin-Ho
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Abstract
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Site-directed mutagenesis of the Bacillus macerans cyclodextrin glucanotransferase (cgt) gene was performed using a modified method of the megaprimer polymerase chain reaction. Relatively longer PCR products were synthesized between 816 and 1,211 bps using mis-matched internal primer during the first round reaction, and these megaprimers were used to successfully amplify the cgt gene. To avoid a sequence-shift caused by non-specific addition of adenine at 3´ end of PCR product, a primer was designed. Even with the constraint, this method extends the PCR technique in producing internal or multiple site mutations.
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