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Schematic diagram of a motif-mixing protocol used in this study

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posted on 2011-12-30, 18:10 authored by Hirohide Saito, Tamiko Minamisawa, Kiyotaka Shiba

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Taken from "Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs"

Nucleic Acids Research 2007;35(6):e38-e38.

Published online 7 Feb 2007

PMCID:PMC1874597.

© 2007 The Author(s)

Initially, we designed DNA sequences for microgenes that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes. These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A and B, while motifs C and D are in the antisense primers, producing primers C and D. The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and dNTP. The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.

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