Zolotukhin S B, Ishchenko I D, Staverskaia O V, Ryndich A V, Kavsan V M
Mol Biol (Mosk). 1982 Jan-Feb;16(1):47-54.
cDNA synthesized on rabbit bone marrow erythroid cells pre-mRNA was cloned in bacterial plasmids. Cold phenol extracted pre-mRNA was a several times more effective template in the reaction of reverse transcription without oligo(dT) 10-primer than hot phenol extracted pre-mRNA. There was no yield increase of DNA-product on hot phenol extracted pre-mRNA in the reaction of reverse transcription with the oligo(dT)10-primer addition. The "hot phenol" poly (A)+-pre-mRNA was used to obtain the representative, full-sized cDNA. The double-stranded form of this cDNA, obtained with the help of DNA-polymerase I, was inserted into the PstI-site of pBR322 plasmid. About 25% E. coli JC5183 clones, transformed with this hybrid plasmid, were found to contain the globin sequences.
在兔骨髓红系细胞上合成的cDNA前体mRNA被克隆到细菌质粒中。冷酚提取的前体mRNA在无寡聚(dT)10引物的逆转录反应中作为模板比热酚提取的前体mRNA有效几倍。在添加寡聚(dT)10引物的逆转录反应中,热酚提取的前体mRNA上的DNA产物产量没有增加。“热酚”聚(A)+前体mRNA用于获得代表性的全长cDNA。借助DNA聚合酶I获得的这种cDNA的双链形式被插入到pBR322质粒的PstI位点。用这种杂交质粒转化的约25%大肠杆菌JC5183克隆被发现含有珠蛋白序列。