López-Cánovas Lilia, Sánchez-Alonso Axel, Higginson David, Ariosa Concepcion, Clark Hilda, Riverón Ana Maria
Molecular Biology Department, Cuban Center for Neuroscience, National Center for Scientific Research, Havana, Cuba.
Electrophoresis. 2003 Apr;24(7-8):1152-8. doi: 10.1002/elps.200390148.
The fastest protocol for Pseudomonas aeruginosa subtyping by contour clamped homogeneous electric field (CHEF) electrophoresis takes around 20 h. It includes enzymatic sample preparation, DNA restriction and fragment separation. Here, P. aeruginosa cells embedded in agarose miniplugs were lysed and deproteinized by incubating the miniplugs for 30 min in a single nonenzymatic solution. DNA molecules were digested for 2 h with 5 U of XbaI, and fragments were separated in 4.96 h by miniCHEF electrophoresis at 10 V/cm. Total time for P. aeruginosa subtyping was 8 h. Control experiments included DNA preparation by enzymatic or nonenzymatic protocols, different times of DNA restriction and comparisons of DNA separations done by miniCHEF or CHEF electrophoresis. Both methods and chambers gave similar results, but the rapid nonenzymatic method and the miniCHEF gave them in less time. Cells grown in broth or on plates were useful for nonenzymatic DNA preparation. Thirteen P. aeruginosa isolates were successfully fingerprinted using the protocol described here.
通过轮廓钳位均匀电场（CHEF）电泳对铜绿假单胞菌进行亚型分型的最快方案大约需要20小时。它包括酶促样品制备、DNA限制酶切和片段分离。在此，将嵌入琼脂糖小胶块中的铜绿假单胞菌细胞在单一非酶溶液中孵育30分钟，使其裂解并脱蛋白。用5 U的XbaI对DNA分子进行2小时的酶切，片段通过在10 V/cm下的miniCHEF电泳在4.96小时内分离。铜绿假单胞菌亚型分型的总时间为8小时。对照实验包括通过酶促或非酶促方案进行DNA制备、不同的DNA限制酶切时间以及miniCHEF或CHEF电泳进行的DNA分离比较。两种方法和电泳槽都给出了相似的结果，但快速非酶促方法和miniCHEF在更短的时间内完成。在肉汤或平板上生长的细胞可用于非酶促DNA制备。使用此处描述的方案成功地对13株铜绿假单胞菌分离株进行了指纹图谱分析。
