Often, less frequent mutations are not found because the new methods of mutational analysis are more sensitive but only for the identification of specific known mutations 9, 28, 29 or because the same exons of a gene are not always investigated in different studies.
Methods: For mutational analysis of the IgE-binding epitopes, peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single or multiple AA substitutions.
However, current methods of mutational analysis are limited to population level luciferase assays in transformed cell lines or qualitative analysis in transgenic embryos.
In this study, EGFR TMDA showed to be a robust method for mutational analysis since no reaction failure was observed in the analyzed samples.
False-negative results accounted for 85% of all the genotype errors made in the scheme and might be due to the low sensitivity of the method used for mutational analysis.
Our approach is similar to that described by Normanno et al (2013) as it reduces the inter-laboratory variability related to the type of technique used for the dissection and allows a comparative evaluation of the sensitivity and specificity of the methods used for the mutational analysis.
Furthermore, a single, standardized method to perform the mutational analysis is not yet available [ 8], making rigorous quality control tests mandatory for each laboratory.
Next generation sequencing is helping to overcome limitations in organisms less accessible to classical or reverse genetic methods by facilitating whole genome mutational analysis studies.
Relative folding free energies determined from GB1-phage sequence data agree well with published GB1 thermal stability studies, validating the use of phage display to conduct quantitative stability studies on GB1, and further suggesting that this method is generally applicable to mutational analysis of protein stability.
While experimental methods such as loss of function mutational analysis, RNAi, or targeted misexpression approaches have been very successful in identifying protein functions, they are labor intensive and time consuming.
