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Since L418A mutation site is structurally far from the active site, it cannot influence the binding of the substrate directly.

In contrast, the Glu196Lys mutation site is buried as part of catalytic domain α-helix in protein core.

Note in the 1 × 1, 2 × 1 and 2 × 2 sequences, the nucleotide at the mutation site is complementary to that of the wild-type sequence.

This means that the fraction of non-synonymous SNPs covering a correlated mutation site is smaller for the interacting pairs (8.4%) than for the non-interacting pairs (11.3%).

As the mutation site is immediately followed by NGR motif, we next tested whether the mutant endostatin has aminopeptidase N inhibitory activity.

One potential drug-resistant mutation site is the 'gatekeeper' residue Thr701; indeed the corresponding gatekeeper mutation in BCR-ABL, PDGF, and c-KIT has been demonstrated to confer resistance to type II inhibitors such as imatinib and nilotinib.

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Thirty primers specific to each potential mutation site were designed to yield SBE products with sufficient mass differences.

Sequences surrounding these 10 sites and integrated −28 (A>G) mutation site were PCR amplified and deep sequenced using IlluminaHiseq 2500 PE150 as paired-end 150 reads.

A saturation mutagenesis library at site G276 was constructed, and the mutation site was designed using the method of NNK degeneracy (N, adenine/cytosine/guanine/thymine; K, guanine/thymine) to encode all 20 amino acids (Tian et al. 2013).

Unexpectedly, no SNPs in relation to this mutation site are presented in the database.

No Hawaiian polymorphism was found within D2030 (total 704 recombinants inquired) indicating that the mutation site was nearby or within the cosmid clone D2030.

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