For the R501X mutation detection a 311pb PCR fragment was incubated with 5 units of Nla III restriction enzyme (New England Biolabs®) and the products were resolved by a 3% agarose 1000 (Invitrogen®) or 12% acrylamide gel electrophoresis; normal sequence produces a 204 and 107 pb fragments and the mutant sequence generates the same 107 pb fragment plus additional 128 and 76 pb fragments.
In order to assess the activity specifically mediated by the motif, the sequence was mutated by random nucleotide substitution, and two different mutant sequences were generated, mutant1 (acgctatc) and mutant2 (ctgcacgc).
The concept of quasispecies [ 1] refers to the equilibrium spectrum of closely related mutants, dominated by a master sequence, generated by a mutation-selection process.
These mutants were generated in the EBV-BAC (or Maxi-EBV) system [45] and revertants (where the targeted mutation has been restored to wild-type sequence) generated for each EBV mutant.
The in vitro evolution is required to be conducted as the following "adaptive walk": a single parent sequence generates N mutant sequences as its offsprings, and the fittest individual among the N offsprings will become a new parent in the next generation.
From each aptamer sequence, 63 mutant sequences were able to be generated.
RNAmute, RSRE, and RDMAS evaluate the normalized base-pair distance between an MFE starting structure and the low energy suboptimal structures generated for mutant sequences using the Vienna RNA package [ 34, 35, 39].
The mutant RNA sequences were generated starting from the sequences of Ang2 aptamers published in the previous literatures [ 13, 14].
A protocol was recently developed for C. elegans that combines WGS with single nucleotide polymorphism (SNP) mapping to simultaneously map and sequence mutants generated by ethylmethanesulfonate (EMS) mutagenesis (Doitsidou et al. 2010).
Using the pLenti-CMV-DEST Gateway Vector (w118-1), we subcloned the wild-type (WT) or mutant coding sequences and generated lentiviruses using a Third Generation Packaging System in 293 T cells.
