Barton B M, Harding G P, Zuccarelli A J
Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, California 92350, USA.
Anal Biochem. 1995 Apr 10;226(2):235-40. doi: 10.1006/abio.1995.1220.
We have devised a method for detecting and estimating the sizes of large bacterial plasmids in the presence of genomic DNA by pulsed-field gel electrophoresis (PFGE). Bacteria harboring plasmids were embedded in agarose and lysed using a rapid protocol. Plugs were incubated with S1 nuclease and subjected to PFGE in agarose gels. S1 nuclease converted supercoiled plasmids into full-length linear molecules. Large plasmids migrated as discrete bands that were readily observed after ethidium staining. Their sizes were reliably estimated by comparison with linear DNA markers. Without S1 digestion, supercoiled plasmids migrated at rates that were not a simple function of their molecular weights, making size determinations problematic. S1-PFGE detected megaplasmids up to 609 kilobases (kb) in six genera of bacteria (Agrobacterium, Escherichia, Klebsiella, Pseudomonas, Salmonella, and Staphylococcus). The procedure gave size values consistent with previous estimates for characterized megaplasmids. Eight new plasmids between 102 and 316 kb were discovered in Klebsiella and Staphylococcus. S1-PFGE avoids the difficulties of plasmid isolation, eliminates the preparation of probes, and does not require knowledge of restriction enzyme cleavage sites. It detects multiple large plasmids up to the limits of PFGE and can be used to screen for megaplasmids in many strains simultaneously.
我们设计了一种通过脉冲场凝胶电泳（PFGE）在基因组DNA存在的情况下检测和估计大型细菌质粒大小的方法。将携带质粒的细菌包埋在琼脂糖中，并使用快速方案进行裂解。将琼脂糖块与S1核酸酶一起孵育，然后在琼脂糖凝胶中进行PFGE。S1核酸酶将超螺旋质粒转化为全长线性分子。大型质粒以离散条带的形式迁移，在溴化乙锭染色后很容易观察到。通过与线性DNA标记物比较，可以可靠地估计它们的大小。如果不进行S1消化，超螺旋质粒的迁移速率与其分子量并非简单的函数关系，这使得大小测定存在问题。S1-PFGE在六个细菌属（农杆菌属、大肠杆菌属、克雷伯菌属、假单胞菌属、沙门氏菌属和葡萄球菌属）中检测到了高达609千碱基（kb）的大质粒。该方法得到的大小值与先前对已鉴定大质粒的估计一致。在克雷伯菌属和葡萄球菌属中发现了8个大小在102至316 kb之间的新质粒。S1-PFGE避免了质粒分离的困难，无需制备探针，也不需要了解限制性内切酶切割位点。它可以检测多个大型质粒，直至PFGE的检测极限，并且可用于同时筛选许多菌株中的大质粒。
