JOURNAL OF 法：IENCE & ENGINEERING MAY, 1967 VOL JV PP. 185~ 190

Preparation of Ribonucle Acids and Deoxyribonucleic

Acids from Fish Livers

by Hw。ang Hwa Yang

Part Two Assoeiate Professor, Department of Chemistry,

Taiwan Provincial Chung Hsing University,

In primary communication (1), we have dealt with the extraction of nucleic acid

frcm fish egg.

Pre盟nt paper reports some results on the preparation of ribonucleic acids (RNA)

-and dexyribonuc1eic acid (DNA) from fish livers by simple procedure and common

reagents.

Experimental

Preparation

The fish livers serving as the starting materials had been obtained from the

market of taichung. Dogfish liver and liver of giant giant black marlin were u揖d

for preparation 1, and prepararation 2, respectively.

Precedures for preparation 1.

2.5 kg of dogfish liver was suspended in 21 of 0.14 M NaCl solution rep扭扭nting

a modification of the one employed by Grinnan and Mosher (2, 3). After extraction

for several hrs the suspension was pas記d through muslin, the sediment was

distributed in 11 of 10% NaCl solution for extraction of DNA representing a

modification of the one employed by Chargafff, Liphshitz and Green (4) and was

passed through muslin. The addltlion of twice their volume of 95% ethanol

produced copious sediments (undissociaed lipid-protein-nucleic acid complex)

Extraction of RNA

Added to RNA sediment 11 of 0.14 M Nacl solution and stirred by Easterin

-electric stirrer for 2 hrs to dissociate lipoidal materials. Addition of 2 times of its

volume of 95% ethanol resulted in lipid-protein-contaminated RNA precipitation and

the following fillraticn removed the majority of the lipoidal contamination. After

addition of 150 ml, of fresh 0.14 M NaCl solution to protein-contami nated RNA

precipitate the solution was shaked for another 10 hrs to breaks hydorogen bonds and

to dissociate the mucleic acid .and the protein During the shaking the solution was

<::overd with a thin layer of chloroform (10 ml) to prevent bacterial gr owth. The

solution was deproteinized by vigorous hand agitation with the addition of another

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27 .5 ml of chloroform at least for 5 minutes. The resulting solution, clarified by

filtration was slowly injected below the surface of 2 volumes of 95% ethanol kept

at -20。C, the fibers cf RNA precipitated out.

Extraction of DNA

Added 200 ml of 10% NaCl solution to DNA sediment and stirred by Eastern

electric stirrer for 2 hrs to dissociate Iipoidal materials. Addition of 2 times its

volume of 95% ethanol resulted in lipid司protein-contaminated DNA sediment and

the fellowing filtration removed the majority of lipoidal contamination. To the

protein-contaminated DNA precipitate added 300 ml of fresh 10 NaCl solution and

shaked bv Cenco-Meizer Sieve Shaker for 5 hrs to dissociate nucleic acid and the

protein.

During thg shaking the solution is covered with a thin layer of chloroform (10

ml) to prevent bacterial growth. The solution was deproteinized by vigorous hand

agitation with another 65 ml of chloroform. The resulting solution, clarified by

filtration was injected below the surface of 2 volumes of 95% ethanol kept at 20°C,

the fiber of DNA precipitated out.

The procedures for preparation 1 are summarized in the figure 1.

Pracedures for preparation 2

1 kg of liver of giant black black marlin suspend in 21 of 0.14 M NaCl solution

for extracton of RNA. After extraction for several hrs the suspension was passed

through muslin, the sediment was distributed in 500 ml of 10% NaCl solution for

extraction of DNA and was passed through muslin.

Extraction of RNA

To the RNA suspension (undissooiated lipid-protein-nucleic acid complex) added

51 (2 times the volumes of RNA suspension) of 95% ethanol and kept for over

night. After filtration added 0.14 M NaCl solution and 2 times its volume of 95%

ethanol. By the following filtration all of the lipids weve completely removed. (The

dogfish liver contains less lipids than the liver of the giant black marlin. After the

protein contaminated RNA precipitate was suspended in 150m/ of 0.14 M NaCl solu­

tion, the solution was deproteinized by viqorous agitation by electric stirrer for

4 hrs, and the sieve shaker for 8 hrs, with 50 ml of chloroform butanol (4:1) solu­

tion. The resulting solution, clarified by filtration was slowly injected below the surf

ace of 2 volumes of 95% ethanol kept at -20。C, the fibers of RNA precipitated out.

Extraction of DNA

With the use of 21 of ethanol and 1096 NaCl solution as described above obtained

the lipid free protein-contaminated DNA precipitate. After addition of 100 ml of

1.0% NaCl solution and 30ml Chloroform to above precipitate and the following

vigorous stirring for 4 hrs and the shaking for 8 hrs, resulted in the deproteinization

of the prepara tion. The resulting solution, clarified by filtration was slowly injected

below the surface of 2 volumes cf ethanol, the fibers of DNA precipitated out.

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Physiologlca.l properti倒

Preparation of Ribonucl;:i Acids and Deoxyribonucleic Acids from Fisb. Livers

Both RNA and DNA are a lustrous, white, non-hygroscopic solids. The results

of colorimetric analysis are summarized in Table 1 and Figure 2.

Table 1. Physidogical properties of RNA and DNA

Biuret test

dipheryl amine test

oricinol test

maximum UV absorption

RNA (-)

（一）

( +) green soln

261 mu

DNA

（一）

( +) blue soln and ppt

（一）

260 mu

Figure 1. The Procedure of the Extraction of the Nucleic Acids from Dogfish Liver

'Dogf岫山rf心gAdded 21 of 0.14 M NaCl for extraction of RNA and 11 of 10臭豆 NaCl for extraction of DNA. Added 2 volumes of 95% ethanol for precipitation and passed muslin.

I Lipid-protein contami叫︱DNA I

Added 200 ml of IQ% NaCl and stirred for 2 hrs. Added 2 vols of ethanol for precipitation and filtered

I Protein c…1叫DNA

Added 300 ml of 10好 NaCl sol­utio且， shaked for 5 hrs. Added 75 mt of chloroform for depro­teinizaUon and filtered

巨︱

Lipid-Protein contaminated RNA

Added 11 of 0.14 M NaCl and stirred for 2 hrs, Added Z vols of ethanol for preci pit­ation and filtered

︱叫…叫剖RNA

Added 150 ml of 0.14 M NaCl solution. Shaked for IO hrs. Added 35 ml of chloroform for deproteinization and filtered

Both DNA and RNA absorb ultraviolet ligat strougly with maximum absorption

about 2260 mµ, falling off to a minimum about 230 to 240 mµ and almost di臼ppearing

at 310 mµ.

Summary

(1) Simple method has been described which permits the isolation of ribonucleic

acid and desoxyribonucleic acid from fish liver or other biological m aterials.

(2) Ribonucleic acids and dexyribonucleic acids were prepared from fresh dogfish

liver and liver of giant black marlin by succesive extractions with 0.14 M NaCl

and 10% NaCl.

(3) Spectrophotometric and colorimetric analysis show the preparations are ribonu­

cleic acid and dexyribonucleic acid.

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n:i.書版第四期

Figure 2. Characterization of Nucleic Acids by Spectrophotometry

1.00

！’，刀川

J

0.9

0.8

0.6

。、 5

0.4

0.2

0.3

310

DNA

RNA

230 250 270 290

Wave length (mµ)

0.1

210

Literature cited

rn Hwoang Hwa Yang: Jouranal of Science and Engineering

(Taiwan Provincial Chung Hsing University)(Under printing)

Edward L. Grinnan and William A Mosher:

]. Biol. Chem. 191, 719 (1951)

Attila Zsndely, Marton Szabolcs, and Bela Tanko:

C.A. 54, 12型8C (1960)

Chargaff, R, Lipshitz, C. Green and 孔1. E. Hodes:

]. Biol. Chern. 192, 223 (1951)

一 188-

~2)

(4)

(3)

Preparation of Ribonucle Ac!ds and Deoxyribonucleic Acids from Fish Liver自

臺戶等魚類內臟之生物化學lL營養化學才兩之研究

第二報關於薑灣魚額肉聽之核酸

第二部 核酸糖核酸及去氧按酸糟核酸之製法

楊 晃 華

村 此報告中敘述自魚肝及其他生物體可得核酸糖核酸及去氧核酸糖核酸之簡便方法。

口故 0.14 M 及 10% 食鹽溶液連續拋出自沙魚及旗魚肝得核酸糖核酸及去氧核酸糖核酸。

目 以此法，抽出之製品經此色法，光電比色法分析結果為核酸糖核酸及去氧核酸糖核酸。

本研究得國家長期發展科學委員會之論助。又研究過程中得化學系劉東昇先生、張世凱、蔣孝

新、陳美江、陳文平問學之幫忙在此衷心感、謝。

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