Cloning and identification of genes in DNA

Now that we have briefly introduced some of the most important tools and techniques of molecularbiology, we can return to the isolation, cloningand identification of genes ofinterest from within prokaryotic genomic DNA or eukaryotic cDNA library sequences. There are two main approaches for this. 

Clearly options (a) and (b) are much preferred since they can give unambiguous identification of a gene of interest within a very small number of clones (possibly one clone) within the clonal library. 

Velvet pressed to master plate and transferred to nitrocellulose filter 

Separate strands by heating, cool, and anneal primers 

There are other important regions in a pDNA expression vector that need to be mentioned as well in order to complete the tour . Foremost is the promoter. Promoters are non-coding 

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It is clear that in this brief overview of molecular biology, we have not covered a number of important areas that have an important impact on the study of metalloproteins. These include molecular cloning and recombinant DNA technology, which allow proteins to be over-expressed and individual amino acids to be mutated to any other of the 19 protein amino acids genome and proteome analysis that enables the sequences of all the genes of the entire organisms to be determined, and the quantification, localization, interactions and, where possible, activities and identification of all of the proteins in an organism,... 

By far, the most widely used application of the yeast two-hybrid system as intimated in Introduction is the identification of protein partners for a test protein of either known or unknown function. Here, the DNA encoding the test protein or the domain of a test protein is cloned in frame into the bait vector. The fish vector contains cDNA and it is constructed so that there is one cDNA molecule per vector. Fish and bait vectors are cotransformed into the appropriate strain of competent yeast, and the resultant transformed yeast cells are screened for growth on SD media and for reporter gene activities. Putative positive clones are then isolated and characterized further. In the next section, each of these stages is discussed in detail. 

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