The competimer oligonucleotide eliminated template-dependent incorrect signal from all reactions regardless of cycling conditions (Figure 3D E), consistent with a model in which template DNA can tether the RCO to bead-bound BCOs in the absence of ligation (Figure 3A).
Although the PCRRC strategy in these two reports is more straightforward, efficient and reliable compared with other PCR-mediated cloning strategies, a restriction enzyme (DpnI) digestion step is still necessary to eliminate template vector as selection for recombinant plasmids.
Addition of actinomycin D to SPT7 reactions reduced the aberrant products in some experiments, but clearly did not eliminate template switching (Fig. 4D).
This approach also eliminates template source-based variation in PCR amplification, a significant cause of assay nonlinearity.
An improved template fabrication process is proposed to eliminate template-induced corrugation.
3) I am still confused by the phrase in the section "SeqZip eliminates template-switching artifacts in the analysis of Dscam1 isoforms": "Although the percentage of total reads aligning to template-switched and unexplained isoforms was low (0.6-0.940.6-0.94% few reads support the majority of Dscam1 isoforms sequenced (99.9%)".
This innovative methodology combines the advantages of template-based placement approaches, due to its fast packing, with the optimization-based ones, presenting floorplan solutions with improved compactability through the complete evolution of the Pareto set, completely eliminating the template setup effort.
10 U of DpnI restriction enzyme (Stratagene) was subsequently added to eliminate phagemid template and incubated for 1 hour at 37°C.
The PCR product was first digested with DpnI at 37° for 4 hr to eliminate the template DNA, and then purified using the Illustra GFX kit (GE Healthcare).
Note that it is important to eliminate the template RNA prior to PDD as DSN can also degrade the DNA strand of RNA DNA hybrids longer than 15 nt [ 23].
Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.
