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Specific base changes were introduced in each of four regions with the potential for base-pairing (stems I-IV), and for each variant sequence, a rate constant for cleavage was determined.
The link to viral mimicry came when Cantor's group found that the same variant sequence is indeed present on corneal cells and also in a herpesvirus protein called UL6.
When the C>T variant sequence was present, the 600 bp PCR fragment was not digested.
A TRF26 double variant sequence containing the P6Y+H12F variations was characterized by SPR.
However, only four epitopes (out of 25) had a mutant variant sequence as the predominant peptide [peptide no.
When the T>G variant sequence was present, the digestion resulted in two fragments of 250 bp and 100 bp.
The variant sequence encoded a proline (P) at this position (A136P) in five of six animals (Fig. 5).
The variant sequence that deviates most from the predicted value is the D4S+P5Y+M7K triple variant.
The optimized quadruple variant sequence (TNF1-opt) has a Kd = 1.6±0.3 µM determined by equilibrium SPR measurements (Figure 4, Figures S5, S6 in File S1).
Notably, a competitor probe containing the variant sequence completely ablates binding of this additional nuclear protein, while an equivalent amount of the wild-type competitor did not.
The stringency and specificity of the hybridisation process was confirmed by inspection of a series of four controls of known single genotype variant sequence.
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