HIGH-EFFICIENCY CLONING OF FULL-LENGTH CDNA
HIGH-EFFICIENCY CLONING OF FULL-LENGTH CDNA
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DOI：
10.1128/mcb.2.2.161
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发表时间：
1982-01-01
影响因子：
5.3
通讯作者：
BERG, P
中科院分区：
文献类型：
--
作者：
OKAYAMA, H;BERG, P
目前广泛使用的互补DNA [cDNA]克隆方法的一个公认的困难是获得包含相应mRNA的整个核苷酸序列的cDNA片段。这里描述的克隆过程减轻了这个缺点。在每微克兔网织细胞mRNA中获得的105个质粒cdna重组中，约10%含有完整的。-或。beta。-珠蛋白mRNA序列，至少30-50%，但很可能更多，包含整个珠蛋白编码区。克隆全长或近全长cDNA的高效率归功于质粒DNA载体[pBR322-SV40重组]本身作为第一链和第二链cDNA合成的引物，产物不需要任何核酸酶处理，并且该过程中的一个步骤导致具有全长cDNA的重组体优先克隆而非具有截断cDNA的重组体。
A widely recognized difficulty of presently used methods for complementary DNA [cDNA] cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 105 plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete .alpha.- or .beta.-globin mRNA sequence and at least 30-50%, but very likely more, contained the entire globin coding regions. The high efficiency of cloning full- or nearly full-length cDNA is attributed to the fact that the plasmid DNA vector [pBR322-SV40 recombinant] itself serves as the primer for 1st- and 2nd-strand cDNA synthesis, the lack of any nuclease treatment of the products, and to the fact that 1 of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA over those with truncated cDNA.