کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2091576 1081739 2010 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification
چکیده انگلیسی

Gene synthesis technologies provide a powerful tool for increasing protein expression through codon optimization and gene modification. Here we describe an improved PCR-based gene synthesis technology, which is accurate, simple and cheap. The improved PCR-based gene synthesis (IPS) method consists of two steps. The first one is the synthesis of 300–400 bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15 bp overlap. The second one is assembling of fragments from the first step into the full-length gene by PCR reaction. Using this approach, we have successfully synthesized a modified phytase gene with 1256 bp in length with optimal codons for expression in Pichia pastoris. P. pastoris strain that expressed the modified phytase gene (phyA-mod) showed a 50% increase in phytase activity level. In addition, we propose an inexpensive method for error correction, based on overlap-extension PCR (OE-PCR).

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Microbiological Methods - Volume 81, Issue 2, May 2010, Pages 147–152
نویسندگان
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