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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Ligation of nonmatching DNA molecule ends.
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Ligation of nonmatching DNA molecule ends.

机译：不匹配的DNA分子的连接终止。

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摘要

T4 DNA ligase can promote the in vitro ligation of blunt DNA ends to ends bearing a 2-nucleotide single-stranded protrusion. This was shown by digestion of plasmids pBR322 and pSP71 with the appropriate restriction enzymes followed by recircularization of the plasmids and transformation of Escherichia coli. It could be ruled out that such nonmatching ligations are due to the presence of contaminating nucleases. The efficiency of ligation is of the same order of magnitude as that obtained with blunt end ligations. The interaction of a number of different combinations of blunt and sticky ends, the latter bearing both 3' and 5' protrusions, was investigated. Ligation of nonmatching ends was shown to take place in all cases. Several ligation junctions were sequenced, showing that during the ligation process the 2-nucleotide protrusion is trimmed away. In two instances the ligation event was accompanied by the specific loss of either 3 or 15 nucleotide pairs as well as the protrusion. An intermolecular ligation involving nonmatching ends was also performed, demonstrating that this form of ligation can be usefully employed in molecular cloning experiments.
机译：T4 DNA连接酶可以促进平端DNA末端到带有2个核苷酸单链突出端的末端的体外连接。通过用适当的限制酶消化质粒pBR322和pSP71，然后将质粒重新环化并转化大肠杆菌，可以证明这一点。可以排除这种不匹配的连接是由于污染性核酸酶的存在。结扎效率与钝端结扎获得的数量级相同。研究了钝头和粘性末端（同时带有3'和5'突起）的许多不同组合的相互作用。显示在所有情况下均发生不匹配末端的连接。数个连接接头被测序，表明在连接过程中2-核苷酸突出被修剪掉。在两种情况下，连接事件伴随着3或15个核苷酸对的特异性丢失以及突出。还进行了涉及不匹配末端的分子间连接，这表明这种连接形式可用于分子克隆实验中。

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