Promoter analysis by saturation muta
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Abstract |
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.
Introduction |
Navigation |
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Methods and Discussion |
Saturation Mutagenesis
Reporter systems
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Oligonucleotide design
Mutagenesis by PCR.
Table 1: Reporter genes used for promoer analysis | ||
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Cloning of the PCR product: library construction
Confirming the randomness of the library
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Analysis Of Mutants
Phenotypic analysis
Transcription analysis
Effects of mutagenesis on transcription
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DNA sequence analysis to identify mutations
Method(s) to derive the consensus sequence
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References |
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