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Confirmation that all the plasmids analysed in this strain had retained the expected mutations and had not undergone further virR changes was obtained after plasmid extraction and sequence analysis.
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This exchange of fragments also well explains the absence of the expected mutations C12705T and G12771A in the haplogroup M7a2 sample.
We also found 90 high-resolution SNPs, including expected mutations in amiE and metZ (Supplementary Figure S1).
For a negative control, wild-type plaques with no expected mutations were collected and processed for sequencing.
Isolates were demonstrated to contain the expected mutations by PCR amplification and nucleotide sequencing of rpoB.
As might be expected, mutations in the ATM and MMR pathways were observed in families where PrCa coaggregated with colon cancer cases (three of the four families), whereas mutations affecting the HR/FA pathway were found mainly in families with breast cancer, and to a lesser extent in families with ovarian cancer in addition to PrCa (six of the families) (Table 2).
In the mutant expression clones, the sequences of the entire region mutated were amplified by PCR and the expected mutations were verified by DNA sequencing.
In both runs, all expected mutations were called in 100 and 50% of diluted DLD1 DNA.
For all H3-L61X strains used for subsequent experiments, sequencing of the corresponding OAD476-OAD477−amplified frevealed revealed the expected mutations at the H3-L61X codon and no additional mutations.
Even though the exons that encode this domain are not enriched for mutations over what was expected, mutations at hotspots R250, R400, and R412 in these exons account for a significant portion (12%) of the mutations in families with VWS/PPS.
As expected, mutations in genes related to Trp metabolism and stress response can be found, but we also observed numerous mutations in the transcription and translation machinery.
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