一种将DNA限制性片段作为单一片段克隆到酵母人工染色体中的方法。

A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes.

作者信息

Sgaramella V, Ferretti L, Damiani G, Sora S

机构信息

Dipartimento di Genetica e Microbiologia A. Buzzati-Traverso, Università, Pavia, Italy.

出版信息

Biochem Int. 1990;20(3):503-10.

PMID:2189412

Abstract

A novel procedure is described for the cloning of partial EcoRI fragments of bovine DNA: it reduces the chance of sequence rearrangements due to multiple insertions (co-cloning) of restriction fragments in the resulting YAC. The DNA to be inserted has been dephosphorylated, whereas the matching ends of the vector, pYAC4, have not. The ligation was essentially complete, the transformation efficiency was close to 19 transformants per ng of vector and the frequency of clones carrying YAC, 60-100 kb in size, was close to 70%. The YACs show segregative and replicative stability.

摘要

本文描述了一种用于克隆牛DNA的部分EcoRI片段的新方法：该方法降低了由于限制片段在所得酵母人工染色体（YAC）中多次插入（共克隆）而导致序列重排的可能性。待插入的DNA已去磷酸化，而载体pYAC4的匹配末端未去磷酸化。连接基本完成，转化效率接近每纳克载体19个转化体，携带大小为60 - 100 kb的YAC的克隆频率接近70%。这些YAC显示出分离稳定性和复制稳定性。