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Plasmid inserts:

My gene, its role, and our successful forward insert

My lab partner, Peter Leese, and I have been working with IDH2 for the past few months and were able to successfully clone our gene in the forward orientation using the pQ30-UA vector. IDH2 has several function as described in the Sacchromyces Genome Database: glutamate biosynthesis
isocitrate metabolism, and the tricarboxylic acid cycle. In the cell, it is localized in the mitochondrial matrix.

Based on previous discussions with Dr.Campbell, it was suggested that the two IDH genes our molecular lab has been working with, IDH1 and IDH2, might be difficult to clone in the forward direction. Although, this hypothesis was, fortuitously, disproved, I am inclined to believe that IDH's biological role could potentially influence the directionality of its insertion.

The main distinction between the IDH and IDP genes is the co-enzyme respective to each. IDH1/2 are NAD-dependent while IDP1/2/3 are NADP-dependent. Because Dr.Campbell was not concerned with the forward direction insertion of IDP, it suggests that NAD-dependence might be a factor to consider when understanding plasmid insertion.

However, my current knowledge of plasmid insertion does not seem complete enough to come to an informed conclusion.

It is interesting to note that many of the IDH genes found on GenBank during the course of this assignment had comments suggesting specific restriction enyzmes for cloning purposes. It is possible that the sensitive cloning nature of this group of genes has been noted and compensated for in other labs.

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