Sensitizing Mutations Exploiting

Exploiting Sensitizing Mutations to Engineer Nucleotide Binding Pockets... 

Acetylcholinesterase inhibition has been widely used for pesticide detection [88-94], but less exploited than protein phosphatase inhibition for cyanobacterial toxin detection. Nevertheless, the anatoxin-a(s) has more inhibition power than most insecticides, as demonstrated by the higher inhibition rates [95]. In order to detect toxin concentrations smaller than usually, mutant enzymes with increased sensitivity were obtained by genetic engineering strategies residue replacement, deletion, insertion and combination of mutations. Modifications close to the active site, located at the bottom of a narrow gorge, made the entrance of the toxin easier and enhanced the sensitivity of the enzyme. 

DDT-R in Drosophila is a useful model system for a number of reasons. DDT was one of the earliest and most widespread pesticides ever used. In addition, individual flies can be readily genotyped for the presence of the resistance-associated mutation using a simple Polymerase Chain Reaction (PCR) based diagnostic that exploits the insertion of the Accord transposable element. This allows us to identify all three genotypes DDT-S/DDT-S (DDT sensitive), DDT-R/ DDT-S and DDT-R/DDT-R (or SS, RS and RR) in individual flies. Recently, in an outstanding example of parallel evolution, insertion of a different transposable element in the 5 end of the Cyp6gl homolog in D. simulans was also shown to be associated with insecticide resistance [8]. DDT-R is therefore a widespread, representative and current mechanism of insecticide resistance. 

In order to exploit the efficiency and sensitivity of DNA-mediated redox reactions in biosensing applications, further improvements in the methodology were required to demonstrate the feasibility of this approach. A practical adaptation of the system would have three features (1) the ability to use a non-cross-linked redox probe, (2) the ability to detect point mutations within oligonucleotides of varying sequence composition, and (3) the ability to achieve in situ hybridization at the electrode surface. The success obtained in each of these areas [25] indicates that this approach may hold promising applications. 

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