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恶性疟原虫大片段DNA在大肠杆菌中克隆不稳定,很可能与恶性疟原虫DNA的A+T含量非常高(82%)有关。酒酿酵母菌与恶性疟原虫的A+T含量较大肠杆菌与恶性疟原虫的A+T接近,因此对100kb以上的恶性疟原虫DNA片段在酵母菌人工染色体(YACs)克隆的稳定性进行了研究。pYAC4经氯化铯标准法纯化,EcoRI和BamHI完全酶切,小牛肠碱性磷酸酶去磷酸化作为载体DNA。恶性疟原虫B8分离株DNA经EcoRI部分酶切,约15μg埋于琼脂糖井内,用EcoRI部分酶切,然后进行脉冲电场梯度凝胶电泳(PFGE),去除<100kb
The large fragment of Plasmodium falciparum clones in E. coli is unstable and is likely to be associated with a very high (82%) A + T content of Plasmodium falciparum DNA. The A + T contents of S. cerevisiae and P. falciparum are similar to those of E. coli and P. falciparum, and therefore the stability of DNA fragments of P. falciparum over 100kb in yeast artificial chromosomes (YACs) was studied . pYAC4 was purified by cesium chloride standard, completely digested with EcoRI and BamHI, and calf intestinal alkaline phosphatase was dephosphorylated as vector DNA. Plasmodium falciparum B8 DNA was partially digested with EcoRI, and about 15 μg was embedded in an agarose well, partially digested with EcoRI, and then subjected to pulsed electric field gradient gel electrophoresis (PFGE) to remove <100 kb