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

 

    1. Retain 5-10 mL DNA for checking DNA quality and progress of digest.
    2. Digest minimum of 20 mg each parent and 40 mg offspring DNA.
    3. Each digest should be 0.2 mg/mL DNA, with 5 U/ug PstI, NEB buffer 3, BSA.
    4. Incubate 37ûC 2hrs.
    5. Add 0.5X volume PstI originally added.
    6. Incubate 37ûC 2 hrs. May be stored indefinitely at Ð20ûC.
    7. Check digest by running 1 mL pre- and 2 mL post-digest DNA on 0.8% gel.
    8. 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

 

    1. 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.

    1. Incubate at 37ûC for 4 hr, spiking reaction with 2.5 mL SAM every hour.
    2. Phenol/chloroform extract; chloroform extract, and NaOAC/EtOH precipitate digests. Dissolve in 100 mL TE. (Can store at 4ûC O/N.)
    3. Quantify DNA concentration by spectrophotometer.
    4. 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

 

    1. 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.
    2. Add 14 mL 5 N NaOH to denature. Mix rapidly. Incubate at RT for 15Õ, mixing every 5Õ.
    3. Neutralize reaction with 29 mL 3M MOPS buffer. pH should be 8.5.  Check with pH paper and adjust if not 8.5.
    4. Split into 2 aliquots, in 1.5 mL screw-cap tubes.
    5. To each tube add:

                                                     i.     32 mL formamide;

                                                      ii.     45 mL H2O;

                                                        iii.     200 mL 2xFPERT buffer.

Mix well.

    1. Add 150 mL phenol to each tube. If solution is clear, continue to add phenol 25 mL at a time until solution becomes cloudy.
    2. Agitate at RT for 12-24 hrs (yeast) or 48 hrs (human).
    3. Extract each tube with 150 mL chloroform. Keep top (aqueous) layer.
    4. Extract with 400 mL chloroform. Keep top (aqueous) layer.
    5. Split combined aqueous fractions into two equal volumes and add 1 mL EtOH to each.
    6. Incubate Ð80ûC 30Õ; spin 4ûC max speed, 30Õ.
    7. 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.

    1. Dissolve each pellet in 45 mL 1xTE. Pool each set of hybrids into 1 tube each.
    2. 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)

 

    1. 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.

    1. Incubate at 37ûC for 1 hr.
    2. Spin down condensation and keep on ice.
    3. Prepare ExoIII mix (per reaction):

                                                     i.     539.4 mL H2O;

                                                      ii.     60 mL 10x ExoIII Buffer;

                                                        iii.     0.6 mL 100 U/mL ExoIII.

    1. Add 600 mL mix to digested heterohybrids. Do this step as quickly as possible.
    2. Incubate 37ûC for 15Õ.
    3. Stop the reaction by adding 8 mL 0.5 M EDTA, 80 mL 8M LiCl to each tube.
    4. Add 30 mL SSAM, well mixed.
    5. Incubate at RT for 10Õ, mixing every 2Õ.
    6. Spin down SSAM in picofuge, 1Õ.
    7. Divide S/N between two 0.45 m Millipore filters.
    8. Spin in microfuge, 1000 rpm, for 3Õ.
    9. Add 1 mL linear acrylamide to each tube and mix well.
    10. Add 900 mL EtOH to each tube and mix well.
    11. Incubate at Ð80ûC for 30Õ.
    12. Spin at maximum RPM, 4ûC, 30Õ.
    13. Wash with 70% EtOH and air-dry pellet.
    14. Resuspend each pellet in 26 mL 1x TE.
    15. Quantify DNA concentration by spectrophotometer.

           

 

F.    Mismatch-specific nicking (MutHLS digest)

 

    1. 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.

 

    1. 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)

 

    1. 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.
    2. Incubate at 37ûC for 10Õ.
    3. Stop the reaction by adding 1 mL 0.5 M EDTA, 5 mL 8M LiCl to each tube.
    4. Add 5 mL SSAM, well mixed.
    5. Incubate at RT for 10Õ, mixing every 2Õ.
    6. Spin down SSAM in picofuge, 1Õ.
    7. Transfer S/N to 0.45 u Millipore filter.
    8. Spin in microfuge, 1000 rpm, for 3Õ.
    9. Add 1 mL linear acrylamide and mix well.
    10. Add 240 mL EtOH and mix well.
    11. Incubate at Ð80ûC for 30Õ.
    12. Spin at maximum RPM, 4ûC, 30Õ.
    13. Wash with 70% EtOH and air-dry pellet.
    14. 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

Phenol, buffer-saturated, pH 7-8

Chloroform

EtOH, absolute and 70%

3M NaOAc

Linear acrylamide

 

 

B.

PstI

 

C.

dam methylase, SAM

DpnI

MboI

 

D.

5 N NaOH

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