Laboratory of Cellular Oncology
National Cancer Institute
Bethesda, Maryland, USA
rev. Jan 2013
Protocol A: Rapid analysis of encapsidated DNA in Optiprep fractions
•This method can be used to quickly analyze encapsidated linear fragments of cellular DNA and encapsidated plasmids (or viral genomes) in their native supercoiled state. In most instances, it is more informative to analyze encapsidated DNA after restriction digestion. Restriction analysis requires use of Protocol B, which removes the proteinase K and SDS (used to liberate encapsidated DNA from the virion).
1) Take 20µl samples of each Optiprep gradient fraction
2) Make a 5x master mix consisting of:
100 mM Tris pH8
100 mM DTT (make fresh, Pierce #20291 “No-Weigh”)
100 mM EDTA
1% SDS
1% proteinase K stock (Qiagen #19131)
3) Add 5 µl of master mix to each sample. Incubate at ~50º C 15 minutes. This digestion step is important for liberating the encapsidated DNA from the virion.
4) Cast a 0.6% TAE/agarose gel. Make sure the casting tray is free of dust. Dust may appear as speckly fluorescence in the imaged gel. The depth of the gel should be fairly thin, such that the DNA sample will entirely fill the well. It is important that the DNA band extend to near the upper surface of the gel. In the gel post-staining step below, a rate-limiting aspect is the speed at which the DNA stain diffuses into the gel. Having the DNA extend to near the surface of the gel results in faster staining and improved band resolution.
•Note: do notpre-cast the gel with DNA staining agents. SDS in the sample can interact with some types of stain (e.g., GelGreen) and this interaction severely distorts DNA migration and shows up as a fast-migrating front of fluorescent SDS micelles.
5) Add 5 µl of Ficoll load dye containing only bromophenol blue (or other fast-migrating dye that won’t obscure ~3-5 kb bands). It’s OK if the sample slightly overflows the well. Electrophorese the gel, then place into a tub with 10 mM Tris pH 8 + 1x staining agent. Use enough volume to completely cover the gel. Exposing the surface of the gel to air during the staining results in swirly-looking background staining. Rock the gel gently for 30-60 minutes. We’ve traditionally used SYBR Green-I (Invitrogen). Two newer dyes, GelRed and GelGreen www.phenixresearch.comalso work well for post-staining. Dyes should be diluted 1:10,000. SYBR Green-I stains relaxed DNA species (nicked-circular or linear DNA) about 5x more effectively than supercoiled DNA. GelGreen and GelRed seem not to discriminate as much between relaxed and supercoiled DNA states.
Protocol B: extraction of encapsidated DNA from Optiprep fractions. It is possible to quickly extract DNA from Optiprep fractions using QIAquick PCR Purification Kit columns (Qiagen). The QIAquick kit cannot be used on crude cell lysates because small DNA fragments in the lysate are in large excess and compete for binding to the silica resin. For analysis of encapdisated DNA in cell lysates, use Protocol C.
1) Digest 10-100µl of Optiprep material with proteinase K (Protocol A, steps 1-3). Follow the instructions for the QIAquick PCR purification kit (Buffer PB, purple columns).
•Important modification: during the drying step (after Buffer PE has been eluted and removed), spin the columns dry for five minutes at room temperature. If the column is spun for only one minute (as the kit instructs), residual ethanol may be eluted into the finished sample. The residual ethanol may make the sample float up out of the loading well and the sample will be lost. On the other hand, spinning the column dry too long can result in the DNA being irreversibly welded to the silica resin. Five minutes of drying is a good compromise.
2) Since the QIAquick cleanup removes the SDS, it’s OK to pre-cast the TAE/agarose gel with GelRed or GelGreen. It’s not important that gels pre-cast with GelRed or GelGreen be thin. However, the relative speed of band migration is somewhat affected by sample loading volume. In other words, if a gel is loaded with 10 µl of marker ladder and 30 µl of test sample, the test sample bands will migrate artificially slowly relative to the marker ladder bands. It’s important to load similar volumes of marker ladder and each test sample. We have no idea what accounts for this puzzling phenomenon.
SYBR Green-I does not work well for pre-cast gels and should only be used for post-staining.
Protocol C: extraction of nuclease-resistant / encapsidated DNA from pseudovirions in cell lysates
In contrast to Protocol B, this old fashioned/traditional method can deal effectively with the vast excess of tiny DNA fragments found in nuclease-digested cell lysate. In this procedure, the digestion reagents are added sequentially, with the goal of destroying the nucleases prior to release of the encapsidated DNA. Even with these precautions, limited amounts of nicking of encapsidated plasmid can occur.
1) Transfer 100 µl of high-salt cell lysate (see main pseudovirus protocol) or Optiprep fraction material into a microcentrifuge tube.
2) Add 10 µl of 0.5 M EDTA. Mix. Add 2.5 µl of proteinase K (Qiagen). Incubate at 37º C 10 minutes.
3) Add 5 µl of 10% SDS. Incubate 10 minutes at 50º C.
4) Transfer digested lysate to a 2ml Phaselock Heavy tube (Brinkmann# 955154045). Add 200µl of equilibrated phenol-chloroform-isoamyl (Roche). Rinse out the lysate tube with 100µl of TE. Combine rinse with lysate in the Phaselock tube. Shake gently for 10 seconds. (Optional: spike the sample with 10 ng of a gel purified 1 kb BspHI fragment of pBluescript (or other 1 kb piece of DNA). This 1 kb spike can be used to monitor the efficiency of DNA recovery).
5) Centrifuge the Phaselock tube 5 min at 15,000 x g.
6) Add 200µl of chloroform:isoamyl (Sigma). It’s OK to add the chloroform right to the same Phaselock tube since the phe-chlor is locked away at the bottom. Shake gently, centrifuge.
7) Transfer aqueous phase to a 1.5ml tube. Add 2 µl of Pellet Paint (Novagen). There should be about 200µl of extracted lysate. Do not add sodium acetate - the lysate already has plenty of salt. Add 2.6 volumes of 95% (190 proof)ethanol*. Precipitate DNA 4ºC overnight (see Crouse & Amorese (1987) Invitrogen/BRL Focus 9(2):3).
8) Allow the samples to warm to room temp. Spin for 60 min at 15,000 x g room temperature.
9) Remove supe carefully using a P1000. Pellet may be flattened and somewhat filamentous.
10) Add 800µl of 70% ethanol (made using 190 proof). Incubate at room temp at least 5 minutes to give salt a chance to dissolve away from the pellet.
11) Centrifuge for 10 minutes. Discard supe, allow pellet to dry.
12) Dissolve pellet in 50µl of TE.
13) Digest ~15 µl of the extracted DNA with a restriction enzyme that will linearize or double cut the encapsidated plasmid (see Notes).
14) Run digested DNA on agarose gel for SYBR Green I staining (see Procedure 4). Compare to known amounts of digested miniprep plasmid and known amounts of 1kb marker fragment. Load a total of 100ng of marker ladder.
•Notes:
•For some HPV pseudovirus types (most notably HPV16) the majority of nuclease-resistant DNA will consist of ~8kb linear fragments of cellular DNA. We believe that these represent loops of cellular DNA protected by proto-capsids present prior to disruption of the cells (see J. Virol 79:2839). To visualize encapsidated plasmids, it is important to treat the extracted DNA with a restriction enzyme that will linearize or fragment the plasmid and also digest the cellular DNA fragments into a manageable <8kb smear. Note that SYBR Green I stains supercoiled DNA poorly, so without restriction enzyme digestion it may be difficult to see supercoiled plasmid over the background of the linear fragments of cellular DNA. It is important to choose a restriction enzyme that is insensitive to mammalian CpG methylation, which may be present on the plasmid, viral, or cellular DNA (see NEB catalog).
•DNA purification must be done on high-salt lysate the described order. Benzonase is a very sturdy enzyme and isn’t completely inactivated by EDTA or SDS. 800 mM NaCl helps to inhibit Benzonase. Extraction of encapsidated DNA from Optiprep-purified capsids is less demanding since the Benzonase has been purified away.
•Using 200 proof (anhydrous) ethanol to precipitate the DNA results in formation of a strange aqueous droplet at the bottom of the tube. The droplet has nothing to do with the phenol or chloroform, since adding 200 proof ethanol to plain DPBS results in droplet formation. Addition of 50 mM EDTA to DPBS makes the problem much worse. 190 proof ethanol or isopropanol don’t cause this mysterious trouble.
•A protocol for extracting DNA from 293TT cells (modified Hirt procedure) can be found here.