Journal: Nature methods
Article Title: Genome-Wide Profiling of Cap-Independent Translation Enhancing Elements in the Human Genome
Figure Lengend Snippet: In vitro selection of RNA elements that mediate cap-independent (CI) translation ( a ) A library of human genomic DNA fragments was inserted into a DNA cassette containing all of the sequence information necessary for mRNA display. For each selection round, the dsDNA pool was in vitro transcribed into ssRNA, conjugated to a DNA-puromycin linker, and translated in vitro . Uncapped mRNA sequences that initiate translation of an intact ORF become covalently linked to a His-6 protein affinity tag encoded in the RNA message. Functional molecules are recovered, reverse transcribed, and amplified by PCR to generate the DNA for the next selection cycle. ( b ) RNA-protein fusion molecules are generated via the natural peptidyl transferase activity of the ribosome. ( c ) Selection progress was monitored by measuring the fraction of S 35 -labeled fusion molecules recovered from the oligo-dT and Ni-NTA affinity columns.
Article Snippet: The mRNA-peptide fusion molecules were purified from the crude lysate using oligo (dT)-cellulose beads (NEB) and reverse transcribed with SuperScriptII (Invitrogen) by extending the DNA primer (5' TTTTTTTTTTTTTTTATCC ACTTCCATGATGATGGT) with dNTPs.
Techniques: In Vitro, Selection, Sequencing, Functional Assay, Amplification, Polymerase Chain Reaction, Generated, Activity Assay, Labeling