Figure 1. Schematic overview of mRNA and cDNA display using
PUREfrex system. The system starts with a designed DNA library
(randomized sequences) harboring T7 promoter and a leader sequence that
is complementary to the DNA tag. DNA library is in vitrotranscribed to mRNA library and undergo ligation with the puromycin-FITC
DNA tag. Tagged product is then gel purified and translated using the
PUREfrex 1.0 kit. The resulting mRNA-peptide conjugate is then gel
purified. The purified product is then either used directly in the
binding step (mRNA display) or first reverse transcribed to become an
mRNA/cDNA-peptide conjugate (cDNA display). Both products are used for
binding against anti-FLAG M2 magnetic beads and the selected conjugates
are further reverse transcribed and sequenced using the Illumina MiSeq
system. Preparation time (hours) for each step is shown in red with
exception of Miseq sequencing (5 days) carried out only after all
samples were obtained.