Shiraishi H, Shimura Y
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Gene. 1988 Apr 29;64(2):313-9. doi: 10.1016/0378-1119(88)90346-0.
We describe a new rapid method for random introduction of single-nucleotide (nt) substitutions into a small segment of cloned DNA. A DNA fragment containing a sequence to be mutagenized is inserted into a multiple cloning site sequence of a vector plasmid. The plasmid is linearized with two adjacent cuts (generating 5' and 3' protruding ends) and then synchronously and unidirectionally digested with exonuclease III (Exo III) so that the 3' termini generated are localized within the target region. A non-complementary alpha-thiophosphate nucleotide is misincorporated into the 3' terminus generated by Exo III. Since the nucleotide analogue is resistant to the 3'-5' exonuclease activity of DNA polymerase I, its misincorporation into the 3' termini is irreversible. Then, the single-stranded region is filled-in with four canonical nucleotides, and the plasmid is recircularized. This procedure was used to mutagenize a specific region of the rnpB gene of E. coli. By sequencing 72 randomly selected clones, we found that 27 clones (37.5%) had nucleotide substitutions distributed within the desired region of a 55-nt-long segment of the gene. The procedure is simple and is applicable to any DNA molecule.
我们描述了一种新的快速方法，可将单核苷酸（nt）取代随机引入克隆DNA的一小段中。将包含待诱变序列的DNA片段插入载体质粒的多克隆位点序列中。用两个相邻的切口将质粒线性化（产生5'和3'突出末端），然后用核酸外切酶III（Exo III）进行同步单向消化，使得产生的3'末端定位在靶区域内。将一个非互补的α-硫代磷酸核苷酸错掺入Exo III产生的3'末端。由于核苷酸类似物对DNA聚合酶I的3'-5'核酸外切酶活性具有抗性，其错掺入3'末端是不可逆的。然后，用四种标准核苷酸填补单链区域，并使质粒重新环化。该程序用于诱变大肠杆菌rnpB基因的特定区域。通过对72个随机选择的克隆进行测序，我们发现27个克隆（37.5%）在该基因55 nt长片段的所需区域内有核苷酸取代。该程序简单，适用于任何DNA分子。
