Pamela J . Cascone and Lawrence M
نویسنده
چکیده
with the thymine residues were isolated and ligated together in a 10-μL reaction mixture (overnight at room temperature) using equal amounts of each fragment. The ligation was terminated by heating to 65°C, and the products were introduced into DH5α E. coli cells. Cells were grown under ampicillin selection and white colonies selected for miniprep isolation. Plasmid DNA was digested with PvuII and analyzed on a 1.5% agarose gel. Those recombinants with inserts greater than the anticipated 400 bp were selected for DNA sequencing using T3 and T7 primers and Sequenase Version 1.0 (Life Technologies). Initial analysis of a subset of the recombinants revealed that one clone contained a poly(A)+ cassette containing approximately 60 adenine residues. The poly(A)+ cassette was liberated from the vector by digestion with BamHI; however, the small size of this fragment (<70 bp) precluded visualization even in 2% agarose. Therefore, we isolated all the DNA in the lane from below 100 bp to the running front (a ladder was used to monitor size and migration). The fragment was then precipitated and introduced into a BamHI site 3′ to the gene of interest. Using this cassette, we were able to study the effects of the poly(A)+ tail on the half-life and translatability of a death-associated message (unpublished). This method could also be used to generate polynucleotide tracts that could be relocated within a DNA to study the effects of sequence-induced structural changes in nucleic acids. REFERENCES
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