Oligo Probe Design for the Gator® GeneSwift Assay Kit
Introduction
Gator Bio’s GeneSwift Assay Kit is a simple, fast, and reliable method to determine the titer and it streamlines a two-step procedure involving both AAV lysis and genome hybridization within a single tube, succeeded by biolayer interferometry (BLI) detection.
Basic Oligo Probe Design Instruction
Pick target sequence(s) anywhere (except ITR) in the insert or for genome integrity from the ends of the insert, e.g. CMV enhancer on one end and SV-40 at the other.
Design one fluorescein-labeled probe and one biotin-labeled probe that hybridizes to the same strand of the target sequence (positive or negative).
Both probes are approximately 35-40nt long and should not overlap.
The fluorescein-labeled probe is labeled only on the 5’ end.
The biotin-labeled probe has an additional 30 Thymines (T) along with biotins added at both 5’ and 3’ ends.
The calculated energy for the probes should be higher than -10 Kcal/mol, this will minimize homo and heterodimer association.
Linguistic sequence complexity (LC%) is a measure of the 'vocabulary richness' of a genetic text based on counting the number of possible combinations of nucleotide combinations ("entropy" of the set of possibilities) to the theoretically possible one. This value for sequence is converted to percentages, 100% being the highest level.
PrimerDigital Probe Designer
A name is not required but preferred for each probe (e.g. Seq1).
Copy and paste your target sequence (e.g. agtcagtcagtcagtcagtc) to the input field. It reads only standard IUPAC nucleic acid code characters and rejects anything else that is low- and upper-case insensitive.
Enter the length of the oligo (e.g. 35-40).
Enter the probe distance that two oligos should separate from each other.
The name and sequence string can be separated with either space or tab, as long as the style is the same for all the probes.
The user can specify individually each sequence location for both probe designs using ‘[‘ and ‘]’ inside each sequence.
Optionally, use two ‘/.../’ signs for the start and end of the excluded region (this is possible multiple times).
Results
This program may report both forward (FWD) and reverse (RVS) oligos for the negative and positive strands of the genome, respectively. They are presented in paired format. It is recommended that a few different pairs of oligos/probes should be made to determine the most optimal pair for the assay.