一种用于在限制性内切酶标记基因组扫描（RLGS）凝胶上克隆斑点DNA的聚合酶链式反应（PCR）介导方法。

A PCR-mediated method for cloning spot DNA on restriction landmark genomic scanning (RLGS) gel.

作者信息

Suzuki H, Kawai J, Taga C, Ozawa N, Watanabe S

机构信息

Shionogi Institute for Medical Science, Shionogi & Co., Ltd., Osaka, Japan.

出版信息

DNA Res. 1994;1(5):245-50. doi: 10.1093/dnares/1.5.245.

DOI:10.1093/dnares/1.5.245

PMID:7584047

Abstract

A PCR-mediated direct cloning for target spot DNA from RLGS gel has been established. The method consists of PCR amplification of adaptor-ligated spot DNA fragments without excluding similar-sized DNA fragments co-localized on RLGS gel, and following selective ligation with the NotI-dT vector. Applying this method, we have successfully cloned several DNA fragments derived from target spots whose intensities change developmentally due to DNA methylation in the telencephalon of C3H/HeN mice. Since only a few micrograms of total DNA is sufficient for our spot cloning, our method may be highly useful when the total DNA sample prepared for cloning is limited.

摘要

已建立一种用于从RLGS凝胶中直接克隆目标斑点DNA的PCR介导方法。该方法包括对连接了接头的斑点DNA片段进行PCR扩增，而不排除共定位在RLGS凝胶上的大小相似的DNA片段，随后与NotI-dT载体进行选择性连接。应用该方法，我们成功克隆了几个源自目标斑点的DNA片段，这些斑点的强度在C3H/HeN小鼠端脑中因DNA甲基化而发生发育性变化。由于仅几微克的总DNA就足以进行我们的斑点克隆，当用于克隆的总DNA样品有限时，我们的方法可能非常有用。