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

Abstract

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.