一种克隆真核生物结构基因序列的通用方法。
A general method for cloning eukaryotic structural gene sequences.
作者信息
Higuchi R, Paddock G V, Wall R, Salser W
出版信息
Proc Natl Acad Sci U S A. 1976 Sep;73(9):3146-50. doi: 10.1073/pnas.73.9.3146.
Complementary DNA, transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded, has been inserted into Escherichia coli plasmids pSC101 and pMB9 by the poly(dT)/poly(dA) "tailing" and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. An estimate of the amount of inserted globin sequences has been provided by fingerprint analysis of globin mRNA sequences hybridized to the purified plasmid chimeras. Inserted sequences so far subjected to detailed analysis have been ascribed to the rabbit beta globin chain. The susceptibility of inserted beta globin, sequences to the restriction endonuclease EcoRI confirms the existence of a site already found through previous nucleotide sequence analysis.
从纯化的兔珠蛋白信使核糖核酸（mRNA）体外转录并使其双链化的互补脱氧核糖核酸（cDNA），已通过聚（dT）/聚（dA）“加尾”和退火技术插入大肠杆菌质粒pSC101和pMB9中。通过珠蛋白RNA与在硝酸纤维素滤膜上原位生长和裂解的克隆的DNA杂交，已证明由该DNA制备物产生的大肠杆菌转化体含有珠蛋白序列。通过对与纯化的质粒嵌合体杂交的珠蛋白mRNA序列进行指纹分析，已对插入的珠蛋白序列的量进行了估计。到目前为止经过详细分析的插入序列已归因于兔β珠蛋白链。插入的β珠蛋白序列对限制性内切酶EcoRI的敏感性证实了通过先前的核苷酸序列分析已经发现的一个位点的存在。
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