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This particular region corresponds to a retrotransposon and is indicative of insertion.
A slight but rapid self-dequenching of 3 was observed, indicative of insertion of the probe into the membrane, whereas no changes were seen for 2 (Supporting Information, Figure S3).
As shown in Fig. 1B (bottom panel), two out of nine clones (clones 3 and 8) were homozygous, as demonstrated by the appearance of the 3.5-kb band (indicative of insertion of the donor DNA in the WAS locus) using WASF1/WASR1 primers pair (see Fig. 1A, bottom).
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Local regions that deviate from an average chromosomal gene-direction can be indicative of insertions, or areas of codon usage bias, or regions of different mutation pressure.
Detection of insertions might be improved by manipulation of the hybridization/wash conditions or changes to the algorithm to specifically identify probe series indicative of insertions.
These breakpoints are clearly indicative of the insertion of additional DNA regions into the chromosome of C. ulcerans BR-AD22.
The additional bands observed in the digested Tg#28zeoR samples are indicative of targeted insertion of the R-Luc cDNA at the TNF-α gene locus.
Finally, using nine sets of primers designed to rps14 and the first exon of sdh2, we failed to recover any PCR products from Carex and Joinvillea indicative of rps14 insertion into the sdh2 intron under conditions in which all three grasses examined (including the basal grass Anomochloa) yielded an sdh2- rps14 product.
As expected for two chromosomes of nearly the same size, there are no large gap regions indicative of significant insertions or deletions.
Discordant paired-end reads, in which one end matches exonic sequence of a gene, but the other end aligns to a region of the genome distinct from the known locus of that gene, are indicative of retrocopy insertions, and can be used to map retrocopies to the genome (Fig. 1B).
On-plate colonies showed Venus fluorescence as well as red fluorescence, indicative of successful gene insertion (Fig. 2C).
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com