Genomic
Mismatch Scanning in Yeast
December 2005
Brown Lab protocol, modified by Maitreya Dunham and Matt
Brauer of the Botstein lab for use with USB enzymes.
A. Preparation
of genomic DNA from yeast (Qiagen protocol) 3-4
days from colony
Large amounts of high-quality genomic DNA are
critical to the success of the procedure.
Yields 100-200 mg
DNA.
B.
Restriction digest of genomic DNA 5-6 hours
- Retain
5-10 mL
DNA for checking DNA quality and progress of digest.
- Digest
minimum of 20 mg each parent and 40 mg
offspring DNA.
- Each
digest should be 0.2 mg/mL DNA, with 5 U/ug PstI, NEB buffer 3, BSA.
- Incubate
37ûC 2hrs.
- Add
0.5X volume PstI originally added.
- Incubate
37ûC 2 hrs. May be stored indefinitely at Ð20ûC.
- Check
digest by running 1 mL pre- and 2 mL
post-digest DNA on 0.8% gel.
- Phenol/chloroform
extract; chloroform extract, and NaOAc/EtOH precipitate digests. Dissolve
in 100 mL
TE. (Can store at 4ûC O/N.)
C.
Methylation of parent (or ancestor) DNA 5 hours minimum
- To
200 mg
DNA add:
i. 100
mL
10x dam methylase buffer;
ii. 2.5
mL
SAM;
iii. H2O
to 900 mL;
iv. 100
mL
dam methylase.
Mix well.
- Incubate
at 37ûC for 4 hr, spiking reaction with 2.5 mL
SAM every hour.
- Phenol/chloroform
extract; chloroform extract, and NaOAC/EtOH precipitate digests. Dissolve
in 100 mL
TE. (Can store at 4ûC O/N.)
- Quantify
DNA concentration by spectrophotometer.
- Perform
MboI, DpnI and Sau3A digests on both methylated and unmethylated DNA to
assess quality/extent of methylation. Sau3A should cut all species;
MboI will cut only unmethylated DNA; DpnI will cut only methylated DNA.
D.
Hybridization (FPERT) 2 days
minimum
- Mix
20 mg
methylated parent 1 with 20 mg offspring and 20 mg
methylated parent 2 with 20 mg
offspring. Bring volume to 200 mL
with water.
- Add
14 mL
5 N NaOH to denature. Mix rapidly. Incubate at RT for 15Õ, mixing every
5Õ.
- Neutralize
reaction with 29 mL 3M MOPS buffer. pH should
be 8.5. Check with pH paper
and adjust if not 8.5.
- Split
into 2 aliquots, in 1.5 mL screw-cap tubes.
- To
each tube add:
i. 32
mL
formamide;
ii. 45
mL
H2O;
iii. 200
mL
2xFPERT buffer.
Mix well.
- Add
150 mL
phenol to each tube. If solution is clear, continue to add phenol 25 mL
at a time until solution becomes cloudy.
- Agitate
at RT for 12-24 hrs (yeast) or 48 hrs (human).
- Extract
each tube with 150 mL chloroform. Keep top
(aqueous) layer.
- Extract
with 400 mL
chloroform. Keep top (aqueous) layer.
- Split
combined aqueous fractions into two equal volumes and add 1 mL EtOH to
each.
- Incubate
Ð80ûC 30Õ; spin 4ûC max speed, 30Õ.
- Carefully
remove S/N. Wash pellet with 70% EtOH and air-dry.
Note: Pellet may be a clear, viscous liquid. If so,
dissolve pellet in 0.5 mL 70% EtOH. Spin max speed10Õ. Repeat 1x or until
viscous pellet is fully dissolved after spin, and all that remains is ÒtypicalÓ
salt/DNA precipitate.
- Dissolve
each pellet in 45 mL 1xTE. Pool each set of
hybrids into 1 tube each.
- Quantify
[DNA] by A260, 1:25 dilution. Spectra have a peak at 230 nm and a shift of the 260
peak upwards.
E.
Selection of heterohybrids (DpnI/MboI/ExoIII digest)
- To
20 mg
each heterohybrid DNA add:
i. 20
mL
10x NEB Buffer 3;
ii. 1x
TE to 195 mL;
iii. 2
mL
20 U/mL
DpnI;
iv. 1.6
mL
25 U/mL
MboI.
Mix well.
- Incubate
at 37ûC for 1 hr.
- Spin
down condensation and keep on ice.
- Prepare
ExoIII mix (per reaction):
i. 539.4
mL
H2O;
ii. 60
mL
10x ExoIII Buffer;
iii. 0.6
mL
100 U/mL
ExoIII.
- Add
600 mL
mix to digested heterohybrids. Do this step as quickly as possible.
- Incubate
37ûC for 15Õ.
- Stop
the reaction by adding 8 mL 0.5 M EDTA, 80 mL
8M LiCl to each tube.
- Add
30 mL
SSAM, well mixed.
- Incubate
at RT for 10Õ, mixing every 2Õ.
- Spin
down SSAM in picofuge, 1Õ.
- Divide
S/N between two 0.45 m Millipore filters.
- Spin
in microfuge, 1000 rpm, for 3Õ.
- Add
1 mL
linear acrylamide to each tube and mix well.
- Add
900 mL
EtOH to each tube and mix well.
- Incubate
at Ð80ûC for 30Õ.
- Spin
at maximum RPM, 4ûC, 30Õ.
- Wash
with 70% EtOH and air-dry pellet.
- Resuspend
each pellet in 26 mL 1x TE.
- Quantify
DNA concentration by spectrophotometer.
F.
Mismatch-specific nicking (MutHLS digest)
- Bring
1 mg
DNA to 25 mL with 1x TE. Add:
i. 5
mL
10x ENH;
ii. 3
mL
PPD*;
iii. 0.5
mL
100 mM ATP;
iv. 10
ng MutH;
v. 100
ng MutL;
vi. 2.5
mg
MutS;
vii. H2O
to 50 mL
total.
*Before using add 2.5 mL 0.1
M DTT and 25 mL
10 mg/mL BSA (not acetylated) to 225 mL PPD.
Also note that the
concentration of MutHLS are dependent on the activity of your enzymes. These concentrations are for use with
USB enzymes and may not be completely optimal.
- Incubate
at RT for 20Õ. Stop the reaction by heating to 80ûC for 5Õ and placing on
ice.
G.
Depletion of nicked duplex (ExoIII digest)
- For
each reaction mix 5 mL 10x ExoIII buffer, 45 mL
H2O and 0.2 mL 100 U/mL
ExoIII. Add to MutHLS-digested DNA.
- Incubate
at 37ûC for 10Õ.
- Stop
the reaction by adding 1 mL 0.5 M EDTA, 5 mL
8M LiCl to each tube.
- Add
5 mL
SSAM, well mixed.
- Incubate
at RT for 10Õ, mixing every 2Õ.
- Spin
down SSAM in picofuge, 1Õ.
- Transfer
S/N to 0.45 u Millipore filter.
- Spin
in microfuge, 1000 rpm, for 3Õ.
- Add
1 mL
linear acrylamide and mix well.
- Add
240 mL
EtOH and mix well.
- Incubate
at Ð80ûC for 30Õ.
- Spin
at maximum RPM, 4ûC, 30Õ.
- Wash
with 70% EtOH and air-dry pellet.
- Resuspend
each pellet in 15 mL 1x TE. Check concentration.
The resulting DNA can be labeled and hybed by your preferred
method. My methods are available
under Protocols on the Dunham Lab website, genomics.princeton.edu/dunham. If you
have trouble getting enough labeled material, you can amplify the DNA. This may increase the noise, though.
Reagents and supplies
General
Chloroform
EtOH, absolute and 70%
3M NaOAc
Linear acrylamide
B.
PstI
C.
dam methylase, SAM
DpnI
MboI
D.
3 M MOPS acid
2xFPERT buffer
|
100 mL
|
4 M Sodium thiocyanate
|
32.4 g
|
20 mM Tris-HCl pH 7.9
|
2 mL 1 M
|
0.2 mM EDTA
|
40 mL 0.5 M
|
filter sterilize
E.
DpnI
MboI
ExoIII
0.5 M EDTA
8 M LiCl
SSAM
F.
MutH
MutL
MutS
PPD (20 mM KPi pH 7.4, 50 mM KCl, 0.1 mM EDTA pH 8)
10xENH (500 mM Hepes pH 8, 200 mM KCl, 40 mM MgCl2,
10 mM DTT)
100 mM ATP
0.1
M DTT
0.2
10 mg/mL BSA (not acetylated)
G.
ExoIII
0.5 M EDTA
8 M LiCl
SSAM