Luk'ianov K A, Luk'ianov S A
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Bioorg Khim. 1997 Nov;23(11):882-7.
An improved version of the in vitro DNA cloning method described earlier is proposed. The method allows amplification by polymerase chain reaction (PCR) of individual DNA molecules of an unknown primary structure followed by sequencing. The modifications described here provide for in vitro cloning by means of a 40-45-cycle PCR (the original protocol required two consecutive amplifications). In addition, the in vitro cloning is suggested to be carried out in special 96-well plates in the presence of ethidium bromide; upon UV irradiation, the wells containing amplified DNA fluoresce to make the analysis of all 96 wells unnecessary. The improved protocol makes the preparation of individual in vitro clones more straightforward and less expensive.
本文提出了一种对先前所述体外DNA克隆方法的改进版本。该方法允许通过聚合酶链反应（PCR）对未知一级结构的单个DNA分子进行扩增，随后进行测序。此处描述的改进提供了通过40 - 45轮PCR进行体外克隆的方法（原始方案需要连续两次扩增）。此外，建议在溴化乙锭存在的情况下于特殊的96孔板中进行体外克隆；经紫外线照射后，含有扩增DNA的孔会发出荧光，从而无需对所有96个孔进行分析。改进后的方案使单个体外克隆的制备更加简便且成本更低。
