5'-TERMINAL SEQUENCES OF EUKARYOTIC MESSENGER-RNA CAN BE CLONED WITH HIGH-EFFICIENCY
5'-TERMINAL SEQUENCES OF EUKARYOTIC MESSENGER-RNA CAN BE CLONED WITH HIGH-EFFICIENCY
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DOI：
10.1093/nar/9.10.2251
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发表时间：
1981-01-01
影响因子：
14.9
通讯作者：
SCHUTZ, G
中科院分区：
文献类型：
--
作者：
LAND, H;GREZ, M;SCHUTZ, G
已经使用了一种用于克隆mRNA的方法，该方法导致高产率的含有完整5′-末端mRNA序列的重组体。它不依赖于自引发来产生双链DNA，因此不需要S1核酸酶消化步骤。相反，cDNA在其3′-末端具有末端脱氧核苷酸转移酶（TdT）的dCMP加尾。第二条链的合成由与3′ -尾杂交的oligo（dG）引发。双链cDNA随后用dCTP加尾并退火至dGMP加尾的载体DNA。用此方法将鸡溶菌酶cDNA克隆到pBR 322中，转化效率为5 × 103个重组克隆/ng ds-cDNA。序列分析显示，19个随机分离的质粒中至少有9个含有完整的5′-非翻译mRNA序列。这些数据有力地支持了溶菌酶mRNA的5′-非翻译区长度不均一的结论。
A method for cloning mRNAs has been used which results in a high yield of recombinants containing complete 5′-terminal mRNA sequences. It is not dependent on self-priming to generate double-stranded DNA and therefore the SI nuclease digestion step is not required. Instead, the cDNA is dCMP-tailed at its 3′-end with terminal deoxynucleotidyl transferase (TdT). The synthesis of the second strand is primed by oligo(dG) hybridized to the 3′ - tail. Double-stranded cDNA is subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. This approach overcomes the loss of the 5′-terminal mRNA sequences and the problem of artifacts which may be introduced into cloned cDNA sequences.Chicken lysozyme cDNA was cloned into pBR322 by this procedure with a transformation efficiency of 5 × 103recombinant clones per ng of ds-cDNA. Sequence analysis revealed that at least nine out of nineteen randomly isolated plasmids contained the entire 5′-untranslated mRNA sequence. The data strongly support the conclusion that the 5′-untranslated region of the lysozyme mRNA is heterogeneous in length.