4.2@ Simple DNA extraction for PCR screening
Introduction
Schween
et al. (2002) reported a simple PCR method to detect transgene from transformed
P. patens. They incubated moss tissue in an extraction
buffer similar to PCR buffer. Extraction in 10 x PCR buffer works as well, and
more than 5 kb can be amplified with LA Taq (Takara) DNA polymerase (Personal
communication from Dr. K. Takechi at Kumamoto University).
Schween et al. (2002) reported that they could
amplify PCR fragments with template DNA extracted from moss grown on minimal
medium, which do not contain ammonium tartrate and glucose, but not from full
medium, which contain ammonium tartrate and glucose unless PVP was added to the
PCR reaction. Young colony within 1 week after inoculation is the best material
(Takechi personal com.). This method is even simpler than the simplified CTAB
method, and yet sufficient for screening of single copy targeted lines. Here, we
would describe our simplest protocol with successful results for screening the targeted
lines of single copy insertion. Note that further simplification may be
possible.
Procedure
1.
Use young colony within 1 week after inoculation.
2.
Dispense 30 µl of 10 x PCR buffer (100 mM Tris [pH 8.4], 500 mM KCl, 15
mM MgCl2) to a 96-well plate.
3.
Pick up a colony with a toothpick. Remove as much agar as possible
4.
Absorb excess water with a paper towel (kim service paper towel)
5.
Squash the tissue in the buffer with the toothpick
6.
Freeze with liquid nitrogen and allow to thaw
7.
Second freeze thaw cycle
8.
Incubate at 68˚C for 10 min
9.
Centrifuge at 5000 rpm for 5 min at room temperature
10.
Prepare the PCR mix excluding PCR buffer: 14.3 µl H2O, 1.6 µl 2.5mM
dNTP, 1 µl each of 10 µM primers, and 0.10 µl of DNA
polymerase (50:1 mixture of recombinant Taq DNA polymerase and KOD plus DNA
polymerase) per tube
11.
Add 2µl of the extracted DNA: As the DNA is extracted in 10 x PCR buffer,
the amount should be exactly one tenth of the reaction volume
12.
Start PCR with an appropriate cycle: ex. 94˚C 2 min pre PCR; 35 cycles of 94˚C 20 sec, 58˚C 20 sec
(annealing), 68˚C 8 min (1 min /(expected
target length/kb)); 68˚C 7 min
Results
20
out of 24 lines showed amplification product either indicating 5' integration
or multicopy insertion in an experiment. 5' integration was 1.4 kb product,
multicopy test yielded product up to 3.5 kb. These lengths are longer than the
0.5 kb product reported by Schween et al.(2002).
Comments
No
PCR product in multicopy test and clear signal in 5' integration test indicate
a high possibility of single copy targeting event. (In this case 5' homologous
region was shorter than 3' homologous region).
When
very young plants are used, PVP nor spermidine is not necessary, even if they
were grown on a medium containing ammonium tartrate and glucose.
We
have not tested if the freeze-thaw cycles improved the extraction efficiency,
or if freeze-thaw is sufficient without incubation at 68˚C. The minimum incubation time for 68˚C incubation was not investigated.
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.