The oligo design tool takes as input two sequences that correspond to the two alleles to be screened (e.g., wild-type/unedited and SNP/edited versions of the genomic target sequence), with each including at least 35 bases on either side of the modification site (i.e., the site at which a researcher is seeking to engineer a single-nucleotide substitution using genome editing technology). The first nucleotide sequence input typically corresponds to the native or wild-type version of the genomic target sequence (i.e., the sequence before editing), while the second nucleotide sequence is identical to the first sequence, except that it includes a single-nucleotide substitution (i.e., the sequence after editing). Once the sequences have been input successfully, clicking the [Submit] button will initiate the oligo design process. The [Reset] button can be used to clear the inputs if needed. An example of this input is shown below:

Please note that the following conditions must be satisfied in order to use the tool successfully:

• Edited input sequences must include exactly one SNP.
• Both wild-type and edited input sequences must be the same length (e.g., if one is 512 bases, the other is also 512).
• Input sequence lengths cannot exceed 1,000 bases.
• Input sequences should consist of at least 35 bases on either side of the SNP site. For optimal probe design, we recommend including ≥50 bases on either side.

Following a successful submission, the oligo design tool will generate a sequence alignment and the recommended sequences for probe and control oligos. Users can return to the input screen by refreshing the page or clicking the [Back] button of the web browser.

Alignment of input sequences

The inputs are first validated to ensure they meet all the requirements mentioned above. Alignment of the sequences is generated with the position of the SNP indicated using standard annotation. An example of this step is shown below:

Probe and control oligos

Sequences of the following five oligos are provided in a ready-to-order format:

• Displacement oligo (presented below in purple with the noncomplementary base indicated by underline)
• Flap-probe oligo - SNP (presented below in green with the 5' fixed sequence indicated by underline and the hexanediol modification indicated by "3C6" in black font)
• Flap-probe oligo - WT (presented below in orange with the 5' fixed sequence indicated by underline and the hexanediol modification indicated by "3C6" in black font)
• SNP control oligo (presented below in blue with the targeted base indicated in red)
• Wild-type control oligo (presented below in blue-green with the targeted base indicated in red)

Depending on the input sequences, the tool will design oligos according to either Design 1 or Design 2 (targeting either the sense or the antisense strand of the genomic target sequence). At the bottom of each output, the tool will indicate which design was employed and present a color-coded diagram indicating the orientations for each design. For more detailed information about the oligo design process for the Guide-it Knockin Screening Kit, please refer to the product user manual. For more insights about the Guide-it Knockin Screening Kit assay, please refer to our FAQs page.

SNP screening assays performed with the Guide-it Knockin Screening Kit can be used to detect the presence of more than one edit in the genomic target region because complete hybridization is required between the assay probe oligos and PCR product to generate a fluorescent signal.

Our oligo design tool for SNP screening assays only allows for the inclusion of one SNP (one nucleotide difference) between the input sequences. However, it can still be used to design assay probes for scenarios in which additional edits are incorporated in the genomic target region (e.g., scenarios when the user wants to engineer multiple nucleotide substitutions in parallel to generate an amino acid substitution or mutate a PAM, etc.). Below we provide an example of how to design assay probes for such scenarios.

Designing an assay to detect two adjacent nucleotide substitutions

Here we demonstrate how to use the oligo design tool to generate assay probes for detecting two adjacent nucleotide substitutions, using the example of an M164W mutation (ATG>TGG) in the PSEN gene:

For this editing scenario, the first base edit starting from the 5' end of the genomic target sequence will be used as a reference to generate the displacement and flap-probe oligos. Here, it would be the A > T substitution (lowercase "t" indicated by the box):

1. Generate the SNP flap-probe oligo and SNP control oligo for detection of the edited sequence and the displacement oligo:
   
   
   1. In the oligo design tool, include the first base edit (A > T substitution, dark blue) in only the second input sequence ("Sequence after editing")
   2. Include the second base edit (T > G substitution, light blue) in both of the input sequences (for both "Sequence before editing" and "Sequence after editing", the input base should be 'g')
   3. Click [Submit] to have the tool design the probes and control oligo
   4. From the output, use the sequences for the displacement oligo, SNP flap-probe oligo, and SNP control oligo to generate oligos
   5. Do not use the sequences for the WT flap-probe oligo or WT control oligo
   

2. Generate the WT flap-probe oligo and WT control oligo for detection of the unedited sequence:
   
   
   1. In the oligo design tool, include the first base edit (A > T substitution, dark blue) in only the second input sequence ("Sequence after editing")
   2. Exclude the second base edit (T > G substitution) from both of the input sequences (for both "Sequence before editing" and "Sequence after editing", the input base should be 'T')
   3. Click [Submit] to have the tool design the probe and control oligo
   4. From the output, use the sequences for the WT flap-probe oligo and WT control oligo to generate oligos
   5. Do not use the sequences for the displacement oligo, SNP flap-probe oligo, or SNP control oligo
   

While we have attempted to make using the oligo design tool as simple and straightforward as possible, users may run into difficulties that are technical or biological in nature. In either case, please reach out to our technical support team for assistance with your designs. Providing additional information such as any error messages encountered and snapshots of the corresponding inputs and outputs will be extremely helpful in these instances. For all support questions, please contact a Technical Support Scientist through our support page or the Live Chat option on the bottom right corner of our web pages.