Screening Assay Design Services

Introduction

It is absolutely essential to develop a robust screening assay that can reliably distinguish between a rare targeted recombination event and the far more frequent random integration of the targeting vector. It is also critical to determine recombination at both the 5' and 3' arm. Depending on the length of the arms, long-range PCR, Southern blot analysis or a combination of both can be used for the initial screen. Irrespective of the method, it is important that the strategy include a probe and at least one primer positioned outside the region of homology. For PCR-based screening, the second primer can be designed to a vector-specific element such as the neo cassette or a loxP site. Targeted recombination at one arm does not necessarily mean that recombination occurred at the other arm and therefore, independent screening strategies must be developed for each arm.

Summary

We recommend establishing screening conditions initially using high quality genomic DNA. While probe specificity and sensitivity can be determined on wild-type DNA, testing PCR primers presents a greater challenge, as the paired primer set will only work following targeted recombination. Therefore, test primer sets can be designed that test the functionality of each individual primer on wild-type genomic DNA. Once assay parameters have been established, primers/probes should be tested with DNA isolated directly from 96-well plates.

CD Genomics has already updated its technology platforms to mainstream NGS, long-read sequencing, and microarray instruments. We continue to strive to offer the same reliable services to pharmaceutical and biotechnology companies, as well as academia and government agencies, for all your sequencing or array needs. If you are interested, please contact us directly for assistance.