3 Results
for term "sites"
- Resources for Functional Genomics Studies in Drosophila melanogaster...genomic, transcriptomic, proteomic, etc. datasets. These include large community collections of y stocks and plasmid clones, meta information sites like FlyBase and FlyMine, and an increasing number of more specialized reagents, databases, and online tools. Here, we introduce key resources useful ~~~
- Genetic and Genomic Tools for the Marine Annelid Platynereis dumerilii...at specic sites in the genome, the frequency of erroneous DNA repair by NHEJ is increased, resulting in insertions or deletions (indels) that generate mutations at the target site. Exogenously provided DNA can be used as a repair template (by, e.g., possessing sequence homologous to the sequence neighboring ~~~
Figure 5Workflow for targeted mutagenesis using TALENs in P. dumerilii. (A) TALENs can be designed to target specific DNA sequences given their modular composition: each repeat in the DNA binding domain contains a repeat variable di-residue (RVD) that recognizes a single nucleotide (blue, HD = C; yellow, NI = A; purple, NG = T; green, NN = G). (i) TALEN pairs are designed to recognize a specified locus using a web-based prediction tool (https://tale-nt.cac.cornell.edu/node/add/talen). (ii) Genotyping PCR of target loci (arrows, primers) to test for SNPs at target site (yellow). (iii) Target sites comprise 15- to 20-bp binding sites for each TALEN separated by a 15- to 16-bp spacer, with unique restriction endonuclease sites in the spacer, to facilitate screening. (B) TALEN expression plasmids are constructed via two-step GoldenGate (Cermak et al. 2011). (C) In vitro cleavage assay to validate that the constructed TALENs are catalytically active against the intended target. TALEN activity is confirmed by the presence of smaller (gray, “cut target”) bands visible by gel electrophoresis (red arrows). (D) Equal amounts of left and right TALEN mRNA are delivered into Platynereis zygotes via micro-injection. (E) Single or small pools of injected larvae are digested with proteinase K to produce a DNA lysate used as template for screening PCR. Genomic DNA from adult worm tail-clip tissue samples are prepared by conventional genomic DNA extraction kits. (F) Mutation screening is performed by PCR amplification of the target locus (yellow) and (i) digestion of the PCR product with the restriction enzyme cutting within the wild-type spacer sequence (jagged line): if mutations are present in the spacer that disrupts the restriction site, some or all of the PCR product will be resistant to digestion, resulting in “uncut” bands. (ii) Larger deletions are detected as smaller PCR bands (red arrow). (G) Subcloning of uncut bands or smaller deletion bands and subsequent sequencing is used to confirm the presence of mutations centered at the target site.