Kennedy W P
Mol Biochem Parasitol. 1984 Jul;12(3):313-25. doi: 10.1016/0166-6851(84)90088-4.
Kinetoplast DNA (kDNA) was extracted from marker isolates of three 'Old World' cutaneous leishmania, L. tropica, L. aethiopica and L. major. Restriction endonuclease digestion followed by electrophoresis showed that each isolate produced a unique pattern of DNA fragments. The kDNA of each isolate was hybridised to Southern blots of digests of all three kDNAs. This showed that the kDNA of each isolate contained sequences unique to that isolate as well as sequences common to all three isolates. The kDNA sequences of L. tropica and L. aethiopica were more closely related than either were to those of L. major. kDNA of each isolate was cloned and plasmids selected which contained a fragment of DNA unique to the isolate from which the kDNA originated. Whole kDNA was hybridised to organisms fixed on a microscope slide. In each case the kDNA hybridised strongly to the kinetoplast of the organism from which the DNA had been extracted. There was a small amount of cross-hybridisation between L. tropica and L. aethiopica confirming the result of the Southern blot hybridisation. The feasibility of a method of isolate identification using recombinant DNA probes containing sequences unique to a particular isolate or group of isolates is discussed.
从三种“旧世界”皮肤利什曼原虫（热带利什曼原虫、埃塞俄比亚利什曼原虫和硕大利什曼原虫）的标记分离株中提取动基体DNA（kDNA）。用限制性内切酶消化后进行电泳，结果显示每个分离株产生独特的DNA片段模式。将每个分离株的kDNA与所有三种kDNA消化产物的Southern印迹杂交。这表明每个分离株的kDNA既包含该分离株特有的序列，也包含所有三个分离株共有的序列。热带利什曼原虫和埃塞俄比亚利什曼原虫的kDNA序列比它们与硕大利什曼原虫的kDNA序列关系更密切。克隆每个分离株的kDNA，并选择含有来自kDNA起源分离株特有的DNA片段的质粒。将完整的kDNA与固定在显微镜载玻片上的生物体杂交。在每种情况下，kDNA都与提取DNA的生物体的动基体强烈杂交。热带利什曼原虫和埃塞俄比亚利什曼原虫之间有少量的交叉杂交，证实了Southern印迹杂交的结果。讨论了使用含有特定分离株或一组分离株特有序列的重组DNA探针进行分离株鉴定方法的可行性。
