Mutational approaches reverse genetics

The Allele-Specific Amplification Assay (ASA) assay is based on the fact that Taq polymerase will not initiate amplification from a primer that has a mismatch at the 3 ends. Two primers are designed so that the 3 base of the primer corresponds to the site of the genetic mutation to be tested, with either the normal or the mutant sequence at the 3 base positions. An unknown sample can then be tested for the presence of the mutation by using both the normal and the mutant primers in PCR with a common reverse primer. If the sample contains only normal sequence, a PCR product will only be produced when the normal primer is used, and similarly when the sample contains mutant sequence a product will only result from use of the mutant primer. Like the PCR-restriction enzyme method discussed, the ASA approach has also been applied to the detection of mutations in the CYP2D6 gene (16). 

This theoretical example of a QSAR assessment is presented to exhibit FDA s current approach to using S AR as a tool in the safety evaluation of substances proposed for use as food contact materials. If anthrafurin (1,6-dihydroxy anthraquinone CASRN 117-12-4 Fig. 7.3) were expected to be an impurity in a food contact material, an immediate initial concern would be raised due to reports in the literature of positive results in the bacterial reverse mutation assay and other in vitro genetic toxicity tests. A literature search did not identify relevant carcinogenicity data for anthrafurin. 

Error correction is thought to occur by stabilizing correct attachments while destabilizing incorrect attachments (41). Experiments in yeast showed that the inhibition of the Ipll/Aurora family of kinases prevents error correction by stabilizing incorrect attachments (38, 42), but how the active kinase corrected attachment errors was not known. This problem was particularly difficult to address because attachment errors are observed infrequently in the presence of active Aurora kinase (43). Experimental approaches that accumulated attachment errors through inhibition of Aurora kinase, for example by genetic mutation (42), did not permit subsequent kinase activation to examine error correction. Reversible small-molecule Aurora kinase inhibitors present a solution to this problem because they can be used to inhibit kinase function and subsequently removed to activate the kinase. 

The chemical genetic approach allowed the interplay between proteins (ShhN, Patched and Smoothened) to be studied using a pair of small molecule tools (robotnikinin and purmorphamine). The study complemented previous work which demonstrated that cyclopamine - a natural product that antagonises Smoothened - could reverse the effects of mutations in Patched. In both cases, the effects of modulating Patched - either indirectly, by targeting ShhN with Robotnikinin, or by mutation - could be reversed by antagonising Smoothened. 

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