In vitro Potassium Permanganate - Primer extension assay (5/6/99)

This method for detecting the paused polymerase in vitro is based on early work by Carey and Gralla.  Baiyong Li showed that that permanganate reactivity approach could detect paused polymerase in our reconstituted transcription reactions.  The transcription reaction is performed with a high amount of extract and a low amount of template.  E.coli DNA is included in the reaction to inhibit nonspecific protein binding, which reduces the proportion of templates that are transcribed.  Since detection of the paused polymerase relies on the permanganate hyper-reactivity of thymines in the transcription bubble, it is essential that transcription occur on a significant proportion of the templates.  For this reason, the amount of a 3 kb template in the reaction is 10 nanograms.  The following write-up was prepared by Larry Benjamin.

On ice, make a master mix of:

Master Mix
0.8 µl/rxn  1 M HEPES-K, pH 7.6
1 µl/rxn 1 µg/µl HaeIII-cut E. coli DNA
1 µl/rxn 10 ng/µl template (~3kb in size)
16 µl/rxn ddH2O (or additional component, such as alpha-amanitin)

In 0.65 ml tubes add 20 µl total of nuclear extract + HEMG0.1 + other protein (in HEMG buffer).

HEMG0.1 = 25 mM Hepes pH 7.6 (KOH adjusted), 12.5 mM MgCl2, 0.1 mM EDTA, 10% Glycerol, 0.1 M KCl.


To these tubes add 18.8 µl master mix.

Add 1.2 µl of desired mixture of 10 mM NTPs in TE (or just TE).  Mix.  Incubate in 21°C water bath for 30' (if there are more than 12 samples, stagger the incubation by 8 minutes per additional set of samples).

Add 5 µl 300 mM KMnO4 (prepare fresh), staggered by 15 s per sample.  Incubate at 21°C for 4'.

Add water to the purple permanganate crystals and rock the tube back and forth for at least 10 minutes at room temperature to insure that the crystals are completely dissolved.  This is very important because the dark color of the solution prevents one from seeing the crystals.  One typically prepares at least several hundred microliters for accurate weighing of the crystals and dispose of the excess at the end of the experiment.
Add 200 µl stop buffer (50 mM EDTA, pH 8, 1% SDS, 0.4 M 2-mercaptoethanol), staggered by 15 s per sample.

Treat additional reaction sets, if necessary.

Add 2 µl 10 mg/ml proteinaseK.  Incubate at 42°C for 30'.

Add 250 µl phenol/CHCl3/IAA.  Mix.  Spin 5'.  Transfer 200 µl supe to 1.7 ml tube containing 20 µl 3 M NaOAc, pH 6.

Add 500 µl 100% EtOH.  Mix.  Incubate at -20°C for at least 15'.  Spin 15'.  Aspirate.  Add 1 ml 75% EtOH.  Invert tube.  Spin 5'.  Aspirate.  Dry pellet at 37°C (must be completely dry).  Dissolve pellets in 35 µl ddH2O.  Transfer to 0.65 ml tube.

Add 65 µl extension mix to samples and to 2 tubes of 35 µl ddH2O.

Extension Mix (per reaction)
2 µl 10 mM dNTP mix
10 µl 10x PCR Buffer*
2 µl 32P-labelled primer (~0.1 pmoles per reaction)
(51-x) µl ddH2O
Mix
x µl Taq polymerase (1.5 Units per reaction when using commercial Taq)

*10X PCR Buffer:

166 mM  (NH4)2SO4
0.67 M  Tris-Cl, pH 8.8
67 mM  MgCl2
0.1 M   2-mercaptoethanol
1 mg/ml BSA
Subject samples to 25 cycles of amplification as follows:
94°C for 1'
52°C for 2'
72°C for 3'
The final extension in the last cycle is lengthened to 10' at 72°C
Add 100 µl phenol/CHCl3/IAA.  Mix.  Spin 5'.  Transfer 95 µl supe to 1.7 ml tubes containing 9.5 µl 3 M NaOAc, pH 6.

Add 250 µl EtOH.  Mix.  Incubate at -20°C for at least 15'.  Spin 15'.  Aspirate.  Add 200 µl 75% EtOH.  Invert.  Spin 5'.  Aspirate.  Dry pellet.  Bring pellet up in 4 µl SLB.

Load samples onto a urea/polyacrylamide gel prerun at 45 W for 20', loading the blank reactions in the side wells.  Run at 45W.  If you wish to fix the gel, cut off the primer band, if present before placing the gel in acetic acid fixative.

Dry the gel.  Phosphorimage or expose to film.