Tsuyoshi Aoyama
Introduction
Forward genetic approach is a useful tool to isolate
genes involved in phenomena of interest. In this chapter, new forward genetic
approach using deletion mutants and a tiling array is described. Irradiation of
ion beam usually induces double strand breaks in the genome. As a result,
deletion mutant lines are generated. The deletion regions are detected by
hybridization of mutant genomic DNA to a wild type genomic DNA probe on a tiling
array.
1.
Irradiation of ion beam
Materials
Esporangium
Note: We usually use dried sporangium.
Procedure
1. Irradiate sporangium with 50~300 Gy of Ne ion beam
(63 keV/µm)
Note: Irradiation procedure is performed at RIKEN
Nishina Center for Accelerator-Based Science.
2.
Isolation of mutants
It is necessary to optimize the methods to isolate
each mutant for any purpose. In this chapter, the basic method to isolate
mutants is introduced.
Materials
Eion
beam irradiated sporangium
EYellow
tips and blue tips for pipetman (autoclave)
EWater
bath
E15
ml round-bottom tube (Falcon, Iwaki, etc)
E
1.5 ml microtube
Solution
EBCDAT+
6 mM Ca (spore germination medium): 1000 ml
H2O |
900 ml |
Stock B |
10 ml |
Stock C |
10 ml |
Stock D |
10 ml |
500 mM Ammonium tartrate |
10 ml (= 5 mM) |
Alternative TES |
1 ml |
CaCl2 2H2O |
0.9 g (= 6 mM) |
Agar |
8 g (= 0.8%) |
|
Fill up to 1000 ml with H2O |
After autoclaving, pour into 9 cm petri dish
Espore
sowing medium: 200 ml
H2O |
150 ml |
Stock B |
2 ml |
Stock C |
2 ml |
Stock D |
2 ml |
500 mM Ammonium tartrate |
2 ml (= 5 mM) |
Alternative TES |
200 µl |
CaCl2 2H2O |
0.18 g (= 6 mM) |
Agar |
1.6 g (= 0.8%) |
|
Fill up to 200 ml with H2O |
Autoclave and store at 45C
ESterilized
water
E10%
Sodium Hypochlorite Solution (Antiformin): Do not autoclave
Procedure
1. Put one sporangia in a 1.5 ml microtube, then add
1ml of 10% Antiformin to sterilize.
2. Mix gently for 5 min and discard supernatant using
pipetman. Usually the sporangium sinks to the bottom of the tube, but be
careful not to discard the sporangium until step 6. Do not touch the sporangium
with a tip of pipetman, otherwise the tiny spores are dispersed in the tube and
it is not possible to recover them.
3. Add 1 ml of sterilized water into the tube and mix
gently for a few minutes.
4. Discard supernatant with a pipetman.
5. Repeat steps 3 and 4 four times.
6. Add 1 ml of sterilized water, crush the sporangium
with the tip of a pipetman (yellow tip), and mix gently. When you crush the
sporangium, you can see the spores disperse in the tube.
7. Dilute the spore solution 100-fold.
8. Add 3ml of spore sowing medium into the 15 ml
round-bottom tube and store at 45C.
9. Add 1 ml of 100-fold diluted spore solution into
the tube.
10. Vortex for 3 seconds and pour onto the spore
germination medium.
11. Incubate at 25C under continuous light for a few
weeks.
12. Transfer a small part of independent colonies to
a new medium and incubate at 25C under continuous light for a few weeks.
13. Observe the phenotype and isolate mutants.
3.
Detection of deletion regions with tiling array
Materials
EGenomic
DNA (mutants and wild type)
EBioPrime
DNA labeling system (Invitrogen)
E
Ppa Tiling Array ver.1
Note: We designed a custom Affymetrix tiling array
covering the whole P. patens genome sequence of approximately 480 Mb at 67}5
bp resolution excluding repeated sequences. The array contains 6,451,867
perfect match probes along with the controls.
Procedure
1. Label the genomic DNA with BioPrime DNA labeling
system according to manufacturefs instruction.
2. Perform tiling array analysis according to manufacturefs
instruction.
3. Analyze the data with TAS and IGB to detect
candidate deletion regions.
Note: This method is still establishing. It is
important to improve the analytic method of tiling array data for detecting
deletion regions efficiently.
Note: After you detect deletion regions, it is
necessary to examine which deletion regions are responsible for mutant
phenotype.