Figure 5.

Enrichment of myc dsDNA vs. pool dsDNA by in vitro selection. (A) Schematic of the selection protocol. Four mixtures of the myc and pool templates were translated in vitro and isolated on dT₂₅ agarose followed by thiopropyl (TP) Sepharose to purify the template fusions from unmodified templates. The mRNA-peptide fusions then were reverse-transcribed to suppress any secondary or tertiary structure present in the templates. Aliquots of each mixture were removed before (B) and after (C) affinity selection, amplified by PCR in the presence of a labeled primer, and digested with a restriction enzyme that cleaves only the myc DNA. The input mixtures of templates were: lane 1, pure myc; lanes 2–4, 1:20, 1:200, and 1:2,000 myc/pool. The unselected material deviates from the input ratios because of preferential translation and reverse transcription of the myc template. The enrichment of the myc template during the selective step was calculated from the change in the pool/myc ratio before and after selection.