• 作者： 林哲志
• 作者服務機構： Molecular Biology Research and Development; Life Technologies; Inc.
• 中文摘要： 這篇文章描述利用細菌野生型的endonuclease A，能簡化單股去氧核醣核酸（ssDNA）的純化，並有效的消除在純化ssDNA過程中雙股去氧核醣核酸的沾染。 由於ssDNA的純化對DNA基因順序的決定（DNA sequencing），製造特定單股DNA的探針，試管基因的突變（In vitro mutagenesis），消除式互補DNA庫（subtractive cDNA library）的建立有決定性的影響，我們發展出一種迅速、快捷的ssDNA純化步驟，能簡化ssDNA的純化，並且消除純化過程中，細菌染色體（chromosomal） DNA和噬菌質粒（phagemid）DNA的污染。而決定這步驟成功與否，是利用細菌體內野生型endonuclease A的特性。在這篇文章，我們利用生化及基因遺傳上的實驗，證明"wild type endonuclease A"是純化ssDNA的主要酵素。
• 英文摘要： With E. coli, large and variable amounts of chromosomal and plasmid DNAs are observed in thesupernatants of overnight cultures when the cells carry an endA mutation, but are not detected by gelelectrophoresis when the cells carry the wild type allele of endA. Significant amounts of nuclease activityin DH11S endA supernatants were detected by two simple assays; the rapid degradation of added pBR322plasmid DNA, as judged by agarose gel electrophoresis, and a decrease of more than 100000 fold in trans-formation efficiency of the added pBR322 plasmid DNA. By employing isogenic endA mutant and wildtype strains of DH11S and DH10B/F' proAB IacI ZΔM15, it was shown that detectable levels of chro-mosomal and plasmid DNAs are observed only in the endA mutant strains. These results indicate thatEndonuclease I activity is responsible for degradation of chromosomal and plasmid DNA usually presentin preparations of ssDNA. Therefore, a wild type endA gene is useful for the rapid and simple productionof highly purified ssDNA from cells containing phagemid vectors.
• 中文關鍵字： DHIIS; ssDNA; endA; phagemid.
• 英文關鍵字： --