SnapDragon is a web-based tool for the design of long dsRNAs for specific gene knockdown in Drosophila melanogaster via an RNAi approach.
Typically, long dsRNAs are made in a two-step process. First, gene-specific primers are used to amplify a gene fragment from a template such as genomic DNA. Second, the PCR fragment is used as a template in an in vitro transcription reaction to make long dsRNA.
What SnapDragon specifically provides is the sequence of one or more appropriate primer pair for PCR amplification of an amplicon that is then used as a template for dsRNA production.
Off-target effects can occur when the dsRNA has sequence similarity to genes other than the target gene. To best control the chance of off-target effects, SnapDragon strictly enforces a base-pair identity threshold for the amplified region (no sequence stretches with X bp or more identity to non-target genes). The default setting is 19 bp (recommended) but that can be adjusted. For information regarding off-target issues, please see Kulkarni et al and Ma et al.
SnapDragon is optimized for the design of long dsRNAs for Drosophila melanogaster. Potentially, the tool could be optimized for design of dsRNAs for use in other organisms (not tested). SnapDragon is not appropriate for mammals since long dsRNAs are problematic in mammalian cells.
Input Form/ Basic Operation
To use SnapDragon, enter a fly gene symbol, CG identifier, or FBgn (upper box) or enter a nucleotide sequence (lower box). For more information about fly genes and their nomenclature, please consult FlyBase.
Default settings based on DRSC experience are provided, but these can be adjusted. More detailed information can be found by clicking the "?" next to each option. Adjustments may be necessary if the selected gene has small exons or many predicted off-targets.
A list of suitable primer pairs will be returned by SnapDragon. SnapDragon will alert you if one or more primer pair matches to multiple regions in the fly genome, as these have the potential to yield additional amplification products if genomic DNA is used as the initial template in the first-step PCR reaction.