December 14, 1972 Dr. F. Kisseljou Biochemistry D.I. Ivanovsky Virology Institute Gaaraleya Street 16 Moscow D98 U.S. S.R. Dear Dr. KiSSeljOu: Dr. Robert MscAllister has informed us of your interest in our techniques for detecting virus-specific nucleotide sequences in cell DNA. sending you a selection of reprints and preprlnts which will hopefully be of assistance. DNA in cells is currently done as follows: I am Our standard assay for Rous sarcoma virus-specific Reagents 7 1) Cell DNA (at least 500 # 1 prepared from at least 5 x 10 cells. 0.02 M EmA, 0.1 M MaCl, lysed with 0.42 SDS and digested overnight at 37O C. with 500 &ml (predigested) pronase. The SDS concentration fs adjusted to 12, and two phenol extractions (gentle shaking at room temperature, no pipetting of DNA - it is poured) are done. of ethanol are added to the aqueous phase, and the sample is stored at -15O C for at least one hour. The DNA is centrifuged at 2000 rpm for 15 minutes, gently resuspended in 9.01 M EDTA, 0.02 M Tris, pH 7.4 after thorough drying, and treated with 100 Ugh1 of pancreatic ribo- nuclease for at least 3hhours (generally overnight) at 37' C. Pkseol extractions (2) are repeated and the DNA is dialyzed for 2-4 days against several Ch8ngeS of 0.1X standard saline-citrate (SSC * 0.15 M NaC1, 0,015 M Ne citrate). and 280 (the 2601280 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 vith a high pressure cell at 50,000 pounds per square inch. (Alternate available methods include depurinationand sonication). The sheared DNA is then ethanol precipitated overnight (after a chloroform extractfon to remove mineral oil (from the pump lind 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. are thanol precipitated overnight and suspended in 0.003 M KIYTA at a final concentration of greater then 5 mg/ml. Cells are suspended (5 x lo6-10 v cells/ml) in 0.05 M Tris HC1, pH 8, Two volumes The optical density is determined at 260 The peak fractions Page 2 Kissel our/Varmus 12-14- 3 2 2) Labeled double stranded viral polymerase product is prepared as described in the aurnuscripts. If only the rapidly reassociating fraction is available due to low polymerase activity, high specific activity DNA (about 2.5 x lo7/& usually labeled with all four deoxynucleotide pre- cursors) is required for detection of a single copy of viral sequences with rearonable quantities (less than 0.5 mg) of cell DNA. If slowly reassociating DNA can be prepared, specific activities of 5 x 10 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). 6 cpm/ug 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. Procedure 1) Reaction mixtures usually contain 400 ccg of cell DNA and 2000 cpm of labeled polymerase product in .003 M EDTA in a final volume of -- 90 bl in a well sealed glass conical tube (Kontes). heated at 100' C for 2 minutes, 10 ul of 4M PB (solution containing 2 M Na2 HPO4 and 2 MNa H2PO4) is added, and the mixture is overlaid wfth mfneral oil and incubated at 68O C. appropriate intervals (e.g., 1,3,8,24,48,72 hours) and placed in 0.01 M PB (2 ml). experiment. Each experiment should include a heterologous cell DNA (e.g. salmon sperm or calf thymus) os a viscosity control. The mixture is Six IS ~1 samples are taken at Samples are kept at 4' C until the conclusion of the 2) Asaavy! are performed with hydroxyapatite elution using 2 ml of a 10 Gut per 50 ml of 0.01 M PB misture of hydroxyapatite. Elutions are performed at 60' C and consist of 3 ml and 2 ml elutions with 0.16 PB and 2 ml and 2ml with p.'*O M PB. .16 M and pooled .40& 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 up, of calf thymus DNA c~.S carrier. OD260 is measured for the pooled 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 condittons described here witk cause at least a doubling of the reassociatimn 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. do not hesitate to write. suwper and would welcome the opportunity to discuss our work further if appropriate financial and travel arrangements could be made. If I can be of further assistance, I plan to be in Europe for a few weeks thfs Yours truly, Harold E. Varmus, M.D. Dewttment of Microbiology HEV: js encl .