Hi worm breeders,
I just did some micro-injection with dsDNA as homologous recombination sequence for CRISPR.
To be safe, PCR product for sequencing longer than the dsDNA injected is better.
I am just wandering if I use the same set of primers for single worm PCR after F2, is the PCR product sequence from the genome or the injected dsDNA?
The injected DNA can stick around indefinitely, in the form of an extrachromosomal array. It probably won’t - array formation is infrequent, and the array is subject to mitotic loss - but it’s possible. This is why it’s often recommended to include in the injection mix some visible marker (an irrelevant gfp or rfp transgene, for instance) to see whether a transgene formed from the injected DNA is present (and expressing the marker), or only that part of the DNA that recombined homologously into the genome. This is similar to the way that counterselectable markers such as thymidine kinase are used in targeting constructs when doing knockouts in mammalian cell culture.
If you’re worried that the injected DNA construct, still present in an extrachromosomal array (or integrated non-homologously at some inappropriate place in the genome), could give you an erroneous PCR product, because a DNA strand made using it as a template might then act as a primer at the homologous site in the genome, you can test this risk by adding the construct to genomic DNA as template in a control PCR reaction and seeing if such a PCR artifact is possible (though getting the mix just right might be tricky).
Thanks for your help. I’ll try ssDNA as homologous recombination sequence and use the longer primers.