If you isolated a cDNA coding of an unknown gene transcription factor, determine how you could perform the following: (i) isolation of genomic gene encoding for the cDNA and the characterization of the structure, (ii) the chromosomal location of the gene, (iii) and identity of the genes within a cell whose expression is potentially regulated by this transcription factor.
Experiment: Isolate a cDNA of unknown gene transcription factor.
Determine (in detail) the following:
1. isolation of genomic gene encoding for the cDNA and the characterization of the structure
The genomic DNA from the relevant organism is purified and digested with restriction enzymes to generate fragments of DNA. These are then inserted into plasmids digested with the same restriction enzymes, so the genomic DNA fragment ends will be complementary to those in the plasmid. Following ligation, this plasmid preparation is used to transform E. coli bacteria to generate a genomic library, where the whole genome is represented in the bacterial culture. These bacteria are ...
This solution gives a detailed explanation of how, after identifying a novel transcription factor, one would go about isolating the gene encoding this factor, finding the location of this gene on the relevant chromosome, and determining which genes are regulated by this transcription factor.