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.
Procedure
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.
Comments
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).
References
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.