Promoter analysis by saturation mutagenesis.
Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element. This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level. Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.
Institute for Systems Biology. 4225 Roosevelt way NE, Suite 200, Seattle, WA 98105. USA. nbaliga@systemsbiology.org
Nitin S
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Baliga