5′-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency

作者：Hartmut Land, Manuel Grez I, H. Häuser, Werner Lindenmaier, Günther Schütz · 发表于：Nucleic Acids Research · 年份：1981 · DOI：10.1093/nar/9.10.2251 · 被引用次数：366 · 研究领域：RNA Interference and Gene Delivery、Animal Genetics and Reproduction、CRISPR and Genetic Engineering

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 S1 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 x 10(3) recombinant 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.