Deev S M, Karlyshev A V, Polianovskiĭ O L
Genetika. 1982;18(6):880-7.
The copy-DNA was synthesized on the mRNA fraction which was isolated from MOPC 21 mice myeloma by reverse transcription. This copy was completed with another chain without adding the exogenous primer, with the aid of the Klenov fragment of DNA polymerase I. After treatment of the double-stranded pin DNA with endonuclease S1, the poly(dA) sequences were built up using terminal deoxynucleotidyl transferase. The building up of the poly(dT) sequences at the DNA 3'-termini of pBR322 plasmid previously treated with BamHI was performed in a similar way. The average length of connecting polynucleotide sequences was 50 nucleotides. After annealing and transformation of the cells with Escherichia coli hybrid plasmids, selection of clones was made for ApRTcS phenotype. Further, we applied a stepwise selection of clones by means of increasing the specificity: colony hybridization, quantitative hybridization of the excess of plasmid DNA with (32P)-cDNA and hybridization of plasmid DNAs with mRNA units which were transferred from electrophoregrams to the diazo-papers. As a result, bacterial clones were selected which contained gene fragments showing the ability to hybridize with the RNA fraction characterized by mRNA mobility of the light-chain immunoglobulin G.
互补DNA是在从MOPC 21小鼠骨髓瘤中分离出的信使核糖核酸（mRNA）组分上通过逆转录合成的。该互补链在不添加外源引物的情况下，借助DNA聚合酶I的Klenow片段与另一条链完成合成。用核酸酶S1处理双链平端DNA后，使用末端脱氧核苷酸转移酶构建聚（dA）序列。以类似方式在先前用BamHI处理过的pBR322质粒的DNA 3'末端构建聚（dT）序列。连接多核苷酸序列的平均长度为50个核苷酸。用大肠杆菌杂交质粒对细胞进行退火和转化后，选择具有ApRTcS表型的克隆。此外，我们通过提高特异性逐步选择克隆：菌落杂交、过量质粒DNA与（32P）-互补DNA的定量杂交以及质粒DNA与从电泳图谱转移到重氮纸上的mRNA单位的杂交。结果，选择出了含有与以轻链免疫球蛋白G的mRNA迁移率为特征的RNA组分具有杂交能力的基因片段的细菌克隆。
