SnapDragon - Long dsRNA Design

Enter Drosophila Gene Symbol, CG, or FBgn:

Use this field to enter a target gene for dsRNA design. The gene entered can be a fly gene symbol. Examples include wg, hh, RpL21. Alternatively CG identifiers or Flybase Gene identifiers (FBgns) can be used. All of these gene identifiers can be found at FlyBase. Leave this field empty if you have a particular sequence you wish to enter in sequence entry field just below this field.

Or, Copy and Paste Sequence(s)...:

Use this field to enter the nucleotide sequence that the desired dsRNA should target. You can enter RNA or DNA sequence. The sequence may be entered in the form of plain sequence text or in FASTA format. You can enter more than sequence, but multiple sequences must be in FASTA format so that SnapDragon knows where one sequences starts and the other ends. Leave this field blank if a gene identifier is entered in the field above this one.

Select an Off-Target Size:

This field specifies the threshold for predicted off-targets. SnapDragon will avoid predicted off-targets at this specified threshold. Predicted off-targets are determined by searching the fly transcriptome for exact sequence matches to transcripts other than the desired target. The default threshold is 19 bp based on the work of Kulkarni et al. At this length, there is a strong association of predicted off-targets and apparent off-target effects. However, not all 19 bp matches yield an off-target effect and it is possible that matches smaller than 19 bp can (in some circumstances) yield off-target effects. Thus a 19 bp threshold is a guideline and not a hard rule. You will need to use your discretion and judgement.

Select Min Product Size:

This is the minimum length in base pairs of the desired dsRNA.

Select Max Product Size:

This is the maximum length in base pairs of the desired dsRNA.

Select Number of Primer Pairs to Call:

SnapDragon will provide you with primer pair sequences to be used to amplify DNA via PCR to be used as a template for an in vitro transcription reaction to create dsRNA. This field specifies the maximum number of primer pairs that SnapDragon will provide. If a value greater than one is used, you will need to use your judgement as to which primer pair you like the best. SnapDragon will only design primer pairs for dsRNA that meets the other user-specified design criteria. If SnapDragon cannot find such primer pairs, it will not provide any regardless of the specified value of this field.

Select Maximum Primer Pair Penalty (0.0 - 10.0):

To call primers, SnapDragon uses Primer3. The Primer Pair Penalty is a measure of the suitability of the primers for PCR amplification using normal conditions. The lower the number the better. Based on experience, the default is 1.0 which will provide high-quality primers.

Select Forward Primer Endonuclease (optional):

SnapDragon will design a dsRNA that lacks the specified endonuclease restriction site. Additionally, SnapDragon will append the appropriate sequence to the 5 prime primer.

Select Reverse Primer Endonuclease (optional):

SnapDragon will design a dsRNA that lacks the specified endonuclease restriction site. Additionally, SnapDragon will append the appropriate sequence to the 3 prime primer.

Use Advanced Exon Selection:

Use this option if you want to select which exon SnapDragon will use for dsRNA design. If this is unchecked, SnapDragon will decide on the exon for you based on exon size, splice-form universality, and off-target presence. You should check this option if you need to use a specific exon or region of the gene. You should also check this option if SnapDragon has trouble finding a good exon for design. This option has no function if a sequence is pasted into the box; it only applies if a fly gene symbol, CG, or FBgn is provided.

Adjust options as needed:

Use this web page to re-assess and adjust parameters and to decide which exon to use for dsRNA design. By default, the largest exon in the gene is selected. Parameters may need to be adjusted for a variety of possible reasons: For example, the gene may have small exons or a large number of predicted off-targets at the specified threshold.

Please Select an Exon or Exon/Intron Span Below:

Here, you will find a list of exons of the specified gene followed by a list of exons with short intervening introns. They are listed in descending order of length.

Each exon is labeled something like this: FBtr0079432-5/FBtr0079433-4. In this example, this exon is the 5th exon (from the 5' end) of transcript FBtr0079432 and also the 4th exon of transcript FBtr0079433. If the exon is common to all of the gene's transcripts, it will be highlighted and labeled "spliceform universal".

The exons will have predicted off-targets in red. Exon sequence is blue. Intronic sequence (if any) is black. Existing DRSC dsRNAs are underlined.

For Illustration Purposes, a Map of *gene*:

The map image shown here shows the transcripts and existing DRSC dsRNAs for this gene. It links to a more detailed view of the fly genome.

Known DRSC dsRNAs for *gene*:

This is a list of dsRNAs that have been designed at the DRSC. Each dsRNA listed links to more detailed information.

Left

The forward primer to use for PCR amplification.

Right

The reverse primer to use for PCR amplification.

Product Size

The predicted number of nucleotides in the fragment produced by this primer pair.

Pair Penalty (lower is better)

To call primers, SnapDragon uses Primer3. The Primer Pair Penalty is a measure of the suitability of the primers for PCR amplification using normal conditions. The lower the number the better. Higher numbers indicate less suitable primer pairs. If you do not change the Primer Pair Penalty threshold, all primers returned by SnapDragon should be in an acceptable range.

Left Genomic Matches (1 is ideal)

This indicates the number of exact matches of the left primer in the fly genome. In most cases, this number will be 1. If this number is greater than 1, there is some risk that this primer pair will amplify more than one PCR fragment (or the wrong fragment) if genomic DNA is used as template.

Right Genomic Matches (1 is ideal)

This indicates the number of exact matches of the right primer in the fly genome. In most cases, this number will be 1. If this number is greater than 1, there is some risk that this primer pair will amplify more than one PCR fragment (or the wrong fragment) if genomic DNA is used as template.

Left Misprime Penalty (0-10000) (lower is better)

This number indicates the extent to which the left primer partially (as opposed to exactly) matches alternate places in the fly genome. Any score less than 1000 is generally acceptable. (The score is calculated by the following (in this example assume the primer is 20 bp long): For each 16/20 bp match, add 1. For each 17/20 bp match, add 10. For each 18/20 bp match, add 100. For each 19/20 bp match, add 1000.)

Right Misprime Penalty (0-10000) (lower is better)

This number indicates the extent to which the right primer partially (as opposed to exactly) matches alternate places in the fly genome. Any score less than 1000 is generally acceptable. (The score is calculated by the following (in this example assume the primer is 20 bp long): For each 16/20 bp match, add 1. For each 17/20 bp match, add 10. For each 18/20 bp match, add 100. For each 19/20 bp match, add 1000.)

Some Restriction Enzymes (that do not cleave)

This is list of endonucleases whose restriction sites are not found in the predicted PCR fragment. This is not a comprehensive list. This information can be useful for in vivo dsRNAs that require the use of a plasmid vector.