It is necessary to verify the nature of transgene integration in the genome of transgenic P. patens in which unexpected events such as multi-insertion, gene translocation and deletion often occur following transformation. Schween et al. (2002) reported a simple PCR method to detect transgenes from transgenic P. patens. This method is simple, and yet sufficient for screening of transgenic lines. We modified this method for high throughput and call it ggreen PCRh. The key to its success is to prepare fresh samples of young colonies with a green colour. Although green PCR is effective for rapid screening of transgenic P. patens, we recommend carrying out genomic DNA-gel blot analysis of the green PCR-passed transformants, because green PCR alone will not necessarily detect unexpected genome rearrangement by transgenes.
1.Use a young colony within 1 week after inoculation or 3-week-old colonies on selection medium (3rd plate of transformants). DONfT USE samples that have turned brown.
2. Dispense 30 µl of 10 x PCR buffer (100 mM Tris [pH 8.3], 500 mM KCl, 15 mM MgCl2) to a 96-well plate.
3. Pick up a colony with forceps. Remove as much agar as possible
5. Squash the tissue in the buffer with a toothpick or with a TissueLyzer and a 2.3 mm zirconia bead, 20hz, 1min, spindown, twice.
6. Incubate at 68˚C for 10 min
7. Centrifuge at 5000 rpm for 5 min at 4.
8. Prepare the PCR mix excluding PCR buffer: 7.15 µl H2O, 1.2 µl 2.5mM dNTP, 0.75 µl each of 10 µM primers, and 0.075 µl of ExTaq DNA polymerase per tube
9. Add 1.5µl of the extracted DNA
10. Start PCR with an appropriate cycle: ex. 94˚C 3 min pre PCR; 30-40 cycles of 94˚C 30 sec, 55˚C 30 sec (annealing), 72˚C (1 min /(expected target length/kb))
13. Check PCR products by electrophoresis.
Although fragments longer than 5 kb can be amplified with blend Taq (TOYOBO) DNA polymerase PCR, several target sites (including the PIG1 site) could not be amplified with the outward primer set which anneals to genomic DNA outside the homologous targeting region.
The absence of a PCR product in a multicopy test and clear signal in a 5' integration test indicates a high possibility of single copy targeting event. (In this case the 5' homologous region was shorter than 3' homologous region).
Schween, G., S. Fleig, and R. Reski. 2002. High-throughput-PCR screen of 15,000 transgenic Physcomitrella plants. Plant Mol. Biol. Rep. 20:43-47.