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PATRR breakpoint hot spot sequences fold into very long stem-loops [ 7, 10, 20, 21].
Excluding the hot spot sequences themselves from the analysis weakened the associations, but they remained significant for window sizes 4, 10, and 15 through 20 kb.
Finally, alignments of the selected sequences were generated either with clustalw [ 62] and/or cmalign [ 24] in order to better spot sequences that might not be functional.
We have presented a novel bioinformatics tool to rapidly identify RLGS spot sequences by creating a virtual RLGS profile to connect the position of spots with genomic sequences.
The GenBank accession numbers for the human hot spot sequences are: Beta-Globin hot spot: GI 37541814, chromosome 1 hot spots: GI 37549514, and SHOX hot spot: U82668.
Crystal structure interpretation was performed with the aid of MacPyMol http://www.pymol.org/, using crystal structures downloaded from PDB. SIMBAL hot spot sequences were aligned to their own full-length sequences and to the corresponding sequences of proteins with solved crystal structures, and the alignments were inspected manually to confirm accuracy.
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Overall, the agreement between our spot sequencing and the genome traces is extremely high.
Total RNA from the infected P3 CK cells was extracted and analysed by RT-PCR and spot sequence analysis.
Figure 4 shows an example of the vRLGS spot sequence verification experiment.
Results were confirmed by spot sequencing using traditional Sanger methods, as described elsewhere (8 ).
Accurate prediction of a spot sequence was determined if a strong PCR product was obtained when that spot was used as template, but no product or a very weak product was detected using a nearby spot as template.
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