Method for introducing specific and unmarked mutations into the chromosome of Streptococcus pneumoniae
Method for introducing specific and unmarked mutations into the chromosome of Streptococcus pneumoniae
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DOI：
10.1385/mb:26:1:81
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发表时间：
2004-01-01
影响因子：
2.6
通讯作者：
Pozzi, G
中科院分区：
文献类型：
--
作者：
Iannelli, F;Pozzi, G
这项工作描述了在肺炎链球菌染色体中产生位点特异性突变的程序，该程序不涉及使用抗生素抗性标记。通过聚合酶链式反应 (PCR)（通过重叠延伸进行基因剪接）构建转化脱氧核糖核酸 (DNA) 的线性片段，并用于转化肺炎链球菌感受态细胞。通过 PCR 进行转化体的选择，通常，1% 的转化细胞显示出预期的突变。通过该方案，可以将单个碱基对改变为肺炎球菌基因组，以及获得框内删除和插入。
This work describes a procedure for the generation of site specific mutations into the chromosome of Streptococcus pneumoniae that does not involve the use of antibiotic resistance marker. A linear fragment of transforming deoxyribonucleic acid (DNA) is constructed by polymerase chain reaction (PCR) (gene splicing by overlap extension) and used to transform competent cells of S. pneumoniae. Selection of transformants is performed by PCR, and typically, 1% of the transformed cells show the expected mutation. By this protocol it is possible to change a single base pair into pnuemococcal genome, as well as obtaining in frame deletions and insertions.