Dr. Robert MacAllister has informed us of your interest in our techniques for detecting virus-specific nucleotide sequences
in cell DNA. I am sending you a selection of reprints and preprints which will hopefully be of assistance. Our standard
assay for Rous sarcoma virus-specific DNA in cells is currently done as follows:
1) Cell DNA (at least 500 ug) prepared from at least 5 x 10^7 cells. Cells are suspended (5 x l0^6-10^7 cells/ml) in 0.05
M Tris HC1, pH 8, 0.02 M EDTA, 0.1 M NaCl, lysed with 0.4% SDS and digested overnight at 37 degrees C. with 500 ug/ml (predigested)
pronase. The SDS concentration is adjusted to 1%, and two phenol extractions (gentle shaking at room temperature, no pipetting
of DNA - it is poured) are done. Two volumes of ethanol are added to the aqueous phase, and the sample is stored at -15 degrees
C for at least one hour. The DNA is centrifuged at 2000 rpm for 15 minutes, gently resuspended in 0.01 M EDTA, 0.01 M Tris,
pH 7.4 after thorough drying, and treated with 100 ug/ml of pancreatic ribonuclease for at least 3 hours (generally overnight)
at 37 degrees C. Phenol extractions (2) are repeated and the DNA is dialyzed for 2-4 days against several changes of 0.1X
standard saline-citrate (SSC = 0.15 M NaC1, 0.015 M Na citrate). The optical density is determined at 260 and 280 (the 260/280
ratio should be greater than 1.8); if large quantities of DNA have been prepared, part is saved for network formation (to
determine integration of viral genes - see Lepetit manuscript). The remainder is sheared to pieces about 200 nucleotides
in length with a high pressure cell at 50,000 pounds per square inch. (Alternate available methods include depurination and
sonication). The sheared DNA is then ethanol precipitated overnight (after a chloroform extraction to remove mineral oil
(from the pump line) and addition of NaCl to 0.3 M) and (usually) passed over a G-50 sephadex column equilibrated with 0.6
M NaC1, 0.001 M EDTA, and 0.01 M Tris pH 7.4. The peak fractions are ethanol precipitated overnight and suspended in 0.003
M EDTA at a final concentration of greater than 5 mg/ml.
2) Labeled double stranded viral polymerase product is prepared as described in the manuscripts. If only the rapidly reassociating
fraction is available due to low polymerase activity, high specific activity DNA (about 2.5 x l0^7/ug) usually labeled with
all four deoxynucleotide precursors) is required for detection of a single copy of viral sequences with reasonable quantities
(less than 0.5 mg) of cell DNA. If slowly reassociating DNA can be prepared, specific activities of 5 x 10^6 cpm/ug are adequate
due to the greater complexity (high Cotl/2) of this material. (The higher complexity, of course, also indicates that more
sequences are being measured with this DNA).
3) Hydroxyapatite (Biogel-DNA grade) should be standardized for separation of known single and double stranded DNA's in
the presence of the amount of cell DNA present in each sample in a reassociation curve.
1) Reaction mixtures usually contain 400 ug of cell DNA and 2000 cpm of labeled polymerase product in .003 M EDTA in a final
volume of 90 ul in a well sealed glass conical tube (Kontes). The mixture is heated at 100 degrees C for 2 minutes, 10 ul
of 4M PB (solution containing 2 M Na2 HPO4 and 2 M Na H2PO4) is added, and the mixture is overlaid with mineral oil and incubated
at 68 degrees C. Six 15 ul samples are taken at appropriate intervals (e.g., 1,3,8,24,48,72 hours) and placed in 0.01 M PB
(2 ml). Samples are kept at 4 degrees C until the conclusion of the experiment. Each experiment should include a heterologous
cell DNA (e.g. salmon sperm or calf thymus) as a viscosity control.
2) Assays are performed with hydroxyapatite elution using 2 ml of a 10 Gm per 50 ml of 0.01 M PB mixture of hydroxyapatite.
Elutions are performed at 60 degrees C and consist of 3 ml and 2 ml elutions with 0.16 PB and 2 ml and 2ml with 0.40 M PB.
OD260 is measured for the pooled .16 M and pooled .40 M sample to determine reassociation of the cell DNA, and the samples
are then precipitated with a final concentration of 5% trichloracetic acid after addition of 75 ug of calf thymus DNA as carrier.
3) Computations are performed by plotting reassociation of labeled polymerase product against the Cot value for the labeled
DNA. Results are corrected according to single and double-stranded standards run over hydroxyapatite in each experiment.
The conditions described here with cause at least a doubling of the reassociation rate (i.e., 2 fold lowering of the Cotl/2)
if one copy of the viral sequences are present per cell.
Thank you for your interest in our work. If I can be of further assistance, do not hesitate to write. I plan to be in Europe
for a few weeks this summer and would welcome the opportunity to discuss our work further if appropriate financial and travel
arrangements could be made.