Oligo design tool for detection of precise insertions using Guide-it screening assays
Each insert screening assay performed using the Guide-it Knockin Screening Kit involves the application of oligos designed to detect seamless 5' and 3' junctions between the inserted sequence and the surrounding genomic target locus. To simplify the oligo design process, we have developed the software tool below. You will need to input (in the 5'→3' orientation) four sequence fragments corresponding to the 5' and 3' junctions of the inserted sequence and target site following successful editing. (Please see figure below for further clarification.)
- To generate the oligos to detect the 5' junction:
- Enter the wild type sequence 5' upstream of the insertion (1)
- Enter 5' terminal sequence of the insert (2)
- To generate the oligos to detect the 3' junction:
- Enter the 3' terminal sequence of the insert (3)
- Enter the wild type sequence 3' downstream of the insertion (4)
Following submission of the four sequences, the tool will output displacement, flap-probe, wild-type control, and knockin control oligos for analysis of both the 5' and 3' junctions.
NOTE: This tool designs oligo probes to screen for insertions longer than one nucleotide; it does not design probes to screen for single-nucleotide substitutions.
To design oligos for the detection of single-nucleotide substitutions, please use our design tool for SNP screening assays.
Design tool guidelines:
Input sequences should consist of at least 35 bases and cannot exceed 1,000 bases.
NOTE: Please be careful to avoid the inclusion of extra spaces when inputting sequences, as these will be interpreted as part of the input and will likely return an error message.
|5' junction sequences (WT sequence 5' relative to insert, 5' terminal sequence of insert):
5' - ...AACTTTCTATCCGTCCGCGTCAGCGCCTTGCCACCCTCATCTCCAATATGgtgagcaagggcgccgagctgttcaccggcatcgtgcccatcctgatcga...
|3' junction sequences (3' terminal sequence of insert, WT sequence 3' relative to insert):
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