کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2824183 1161627 2011 5 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Enhanced error-prone RCA mutagenesis by concatemer resolution
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی ژنتیک
پیش نمایش صفحه اول مقاله
Enhanced error-prone RCA mutagenesis by concatemer resolution
چکیده انگلیسی

Error-prone rolling circle amplification (RCA) is a promising alternative to error-prone PCR for random mutagenesis. The main disadvantage of error-prone RCA is the low transformation efficiency of the DNA concatemer produced in the amplification reaction. We improved the method by introducing loxP recombination site of bacteriophage P1 Cre recombinase into the target plasmid and reducing the concatemer by Cre recombinase to plasmid-sized units, increasing the number of transformants 50-fold in non-error-prone and 13-fold in error-prone conditions. The efficiency improvement was verified by obtaining 115 ± 57 ceftazidime resistant colonies per recombined RCA reaction from randomly mutated TEM-1 β-lactamase gene library whereas only 9 ± 11 colonies were gained without recombination. Supplementation of the error-prone RCA with Cre/loxP recombination is a simple and useful tool to increase the transformable library size.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Plasmid - Volume 66, Issue 1, October 2011, Pages 47–51
نویسندگان
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