Construction of Disease-Associated ORFs

Wild-type human disease-associated ORFs were selected from the human ORFeome version 8.1 [27 (link)]. As described in Sun et al [17 (link)], human ORFs with disease-associated variants were constructed by site-directed mutagenesis using the Thermo Scientific Phusion Site-Directed Mutagenesis Kit. The Gateway Donor plasmid was amplified using phosphorylated primers that introduce the desired changes followed by a 5-minute, room-temperature ligation reaction. The resulting plasmid was then transformed into NEB5α competent E. coli cells (New England Biolabs).

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Yang F., Sun S., Tan G., Costanzo M., Hill D.E., Vidal M., Andrews B.J., Boone C, & Roth F.P. (2017). Identifying pathogenicity of human variants via paralog-based yeast complementation. PLoS Genetics, 13(5), e1006779.

Publication 2017

Phusion Site-Directed Mutagenesis Kit NEB5α competent E. coli cells

Cells Donor E coli Human Ligation Orfs Plasmid Primers Site directed mutagenesis

Corresponding Organization : Canadian Institute for Advanced Research

Other organizations : Uppsala University, Harvard University

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Variable analysis

independent variables

Site-directed mutagenesis of wild-type human disease-associated ORFs to generate disease-associated variants

dependent variables

Not explicitly mentioned

control variables

Wild-type human disease-associated ORFs
Gateway Donor plasmid amplification using phosphorylated primers
5-minute, room-temperature ligation reaction
Transformation into NEB5α competent E. coli cells

controls

Positive control: Wild-type human disease-associated ORFs
Negative control: Not explicitly mentioned

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