DESOXYRIBONUCLEIC ACID 



97 



DESOXYRIBONUCLEIC ACID 



gation, counted in an hemocytometer 

 chamber, and the Desoxyribonucleic 



Acid (DNA) extracted with hot per- 

 chloric acid and determined by the 

 Dische (diphenylamine) reaction. By 

 this means, the DNA content of a 

 known number of nuclei is determined, 

 making it possible to calculate the av- 

 erage DNA content per nucleus (Boivin, 

 A., Vendrely, R., and Vendrely, C, 

 Compt. rend. Acad, sci., 1948, 226, 

 1061-1063. 



Isolation, Counting, and Washing of 

 Nuclei: Anesthetize the rat, expose ab- 

 dominal and thoracic viscera, clamp 

 the inferior vena cava just below the 

 liver, sever the portal vein, and perfuse 

 the liver through the hepatic vein or 

 thoracic inferior vena cava, using about 

 50 ml. of cold normal saline, followed 

 by 30-50 ml. of cold 2% citric acid. 

 Remove the liver, which will weigh 

 about 10 gm., and dissect away from 

 it any adhering diaphragmatic and con- 

 nective tissues. Frozen liver may be 

 used, but nuclear yield is reduced, and 

 increased cellular debris makes counting 

 difficult. 



The following steps are to be carried 

 out in a cold room at 5°C. After taking 

 a specimen for histological control, ho- 

 mogenize remainder of the liver in about 

 50 ml. cold 2% citric acid (Cunningham, 

 L., GrifRn, C., and Luck, J. M., Gen. 

 Physiol. 1950, 34, 59-63) using a Waring 

 Blendor with metal head equipped with 

 an ice-brine cooling jacket. The cold 

 room alone does not prevent heating, 

 but with a cooling jacket, the tempera- 

 ture should not exceed 5°C. Homogenize 

 for 5 minutes, adding a few drops of 

 capryl alcohol to prevent foaming. 



The yield is 50-70 ml. of a 20% ho- 

 mogenate, which is filtered through four 

 layers of cheesecloth. Microscopic ex- 

 amination at this stage reveals a uni- 

 form suspension of relatively undis- 

 torted nuclei, without any lumped or 

 intact cells. 



Nuclei which are spun down for analy- 

 sis cannot be counted directly, due to 

 clumping, but are estimated by first 

 counting the whole homogenate then 

 subtracting from this value the numbers 

 remaining in the various supernates 

 after centrifugation, as described below. 

 Thoroughly shake the homogenate, 

 withdraw four 5 ml. aliquots, and dilute 

 each with 10 ml. 2% citric acid. These 

 are the counting dilutions, each of 

 which is counted twice in the red cell 

 space (0.1 mm.') of an hemocytometer 

 chamber. Eight to ten individual 

 counts, totaling 3000-5000 nuclei, should 

 keep the error within 5%. 



Fifty ml. graduated conical centrifuge 



tubes are used in the following steps. 

 Remove four 10 ml. homogenate ali- 

 quots (each containing about 

 200,000,000 nuclei as determined above) 

 and carefully layer each over 20 ml. of 

 a solution containing 2% citric acid 

 and 10% sucrose. Centrifuge for 20 

 min. at about 500 x G. By use of this 

 layering technique nuclei are both 

 washed and isolated in a single step 

 (Wilbur, K. M., and Anderson, N. G.. 

 Exp. Cell Res. 1951, 2, 47-57). 



Decant and keep this supernate. Re- 

 wash sediment (containing nuclei) in 

 35 ml. 2% citric acid, centrifuging as 

 above. Combine supernates 1 + 2 from 

 each aliquot, measure volumes, and 

 count nuclei without diluting, if more 

 than 10 per chamber (0.9 mm.^) are 

 present. 



Extraction of DNA: A recent modi- 

 fication of the Schneider procedure is 

 suggested (Schneider, W. C, Hoge- 

 boom, G. H., and Ross, H. E., J. Nat. 

 Cancer Inst., 1950, 10, 977-982). Ex- 

 tract sediment once with cold 12% 

 perchloric acid and twice with cold 6% 

 perchloric acid, centrifuging for 15 min. 

 at 500 X G each time. This removes 

 nucleotides and sucrose. Combine 

 these supernates (3, 4, + 5), measure 

 volumes, and count nuclei as above, 

 adding this value to that of supernates 

 1-1-2, and subtracting the total from 

 that for the whole homogenate. Super- 

 nates 3, 4 + 5 should not be mixed with 

 1 + 2 because cytoplasmic proteins 

 in the latter would be precipitated, with 

 clumping of nuclei. 



Extraction of phospholipids with ab- 

 solute alcohol may be omitted in work- 

 ing with liver and some other tissues. 

 It is necessary for brain. 



Extract sediment with 30 ml. 6% 

 perchloric acid at 90°C. for 15 minutes, 

 cool, and centrifuge. Wash sediment 

 with 10-15 ml. cold 6% perchloric acid 

 and combine these supernates (6 + 

 7) as the DNA extract. This may be 

 stored for 24 hrs. in a deep freeze with 

 little change but longer storage (several 

 weeks) produces unreliable results. 



Determination of DNA: The Dische 

 reagent should be prepared with di- 

 phenylamine that is colorless or nearly 

 so, with a well defined melting point 

 at 53°C. Otherwise this compound 

 must be recrystallized before use. 



Dische Blank 



Reagent Dische 



Glacial acetic acid 100 ml. 100 ml. 



Concentrated H2SO1 2.75 ml. 2.75 ml. 



Diphenylamine 1.0 gm. — 



Following the Seibert procedure (J. 



