Targeted Mutagenesis of TF-binding Motifs

We used the QuikChange site-directed mutagenesis protocol (Agilent Technologies) to mutate individual TF-binding motifs in the RepBase-consensus sequence. We designed primers to target each motif on plasmid constructs and incorporate upto four mutations per motif (Supplementary Tables 6B and 11).
Mutations were introduced at positions in the motif that had the highest information content and were replaced by the least informative nucleotide at that position. We verified the primers to ensure that the mutations did not create a new TF-binding motif, using the TOMTOM tool from the MEME suite of motif analysis67 (link).

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Sundaram V., Choudhary M.N., Pehrsson E., Xing X., Fiore C., Pandey M., Maricque B., Udawatta M., Ngo D., Chen Y., Paguntalan A., Ray T., Hughes A., Cohen B.A, & Wang T. (2017). Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus. Nature Communications, 8, 14550.

Publication 2017

QuikChange site-directed mutagenesis protocol

Consensus sequence Mutations Nucleotide Plasmid Primers Site directed mutagenesis

Corresponding Organization :

Other organizations : Washington University in St. Louis

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Variable analysis

independent variables

TF-binding motifs in the RepBase-consensus sequence

dependent variables

Mutations incorporated per motif

control variables

Positions in the motif that had the highest information content
Mutations that replaced the least informative nucleotide at that position
Primers designed to ensure that the mutations did not create a new TF-binding motif

controls

Positive control: None mentioned
Negative control: None mentioned

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