EXPERIMENT 6 Extraction and Identification of Nucleic Acid from AnimalTissue

1. Purpose

(1) Study and master the principle andoperational techniques of extraction and identification of nucleic acid fromanimal tissue by salting in method.

(2) Know the principle and method ofidentifying nucleic acid qualitatively.

2. Principle

Separate deoxyribonucleoprotein (DNP) from ribonucleoprotein (RNP)according to their difference in solubility in the NaCl solution of definiteconcentration. Then remove protein with denaturant to release nucleic acid. Utilizeundissolvable nature of nucleic acid in ethanol to separate out nucleic acid.At that time we gain our ends to separate and purify.

The solubility of RNP is high and that of DNP is low in 0.14mol/LNaCl solution. The result is contrary in 1mol/L NaCl solution. After the nucleoproteinhave been isolated, we can use protein denaturant (chloroform and isoamylol,sodium dodecyl sulfate (SDS), heated phenol) to remove the protein and releasethe nucleic acid. Nucleic acid will be separated out when add 95% ethanol of1.5~2 times volume to the isolated nucleic acid solution without protein.

Keeplow temperature to inhibit the ribonuclease (RNase) and deoxyribonuclease(DNase) in animal liver, and pay attention to the ions which can activate theenzymes such as Mg²⁺, Fe²⁺ and Co²⁺.

3. Materials

(1) Reagent

① 3,5-dihydroxymethylbenzene (orcinol) reagent: Weight 100mg oforcinol, dissolve in 100ml of concentrated hydrochloric acid, then add 100mg ofFeCl₃•6H₂O.The must be prepared freshly before use.

② Diphenylamine reagent: Weight 1g of diphenylamine (recrystallized),dissolve in 100ml of glacial acetic acid, add 2.75ml of concentrated sulfuricacid and shake up, keep in refrigeratory to standby.

③ 0.14mol/L NaCl solution.

④ 1mo/L NaCl solution.

⑤ 95% ethanol.

⑥ Chloroform.

⑦ Acetone.

⑧ Isoamylol.

(2) Apparatus

① Freezing-centrifuge.

② Ice bath.

③ Tissue triturator or homogenizer.

④ Beaker.

⑤ Measuring cylinder.

⑥ Test tubes.

⑦ Glass stick.

⑧ Scissors.

⑨ Triangular flask or centrifugal tube with stopper.

4. Procedure

(1) Preparing homogenate: Weight fresh orfrozen liver, wash away blood with 0.14mol/L cold NaCl solution (contain0.01mol/L citric acid), cut into pieces with stainless scissors, put into thehomogenizer, add twice volume 0.14mol/L NaCl solution (contain 0.01mol/L citricacid) and prepare homogenate.

Centrifuge the homogenate for 20 minutes (3000rpm). The superstratumis RNP extracting solution and the underlayer is cell fragment and DNP. Removethe supernatant solution to extract RNA. Extract the underlayer twice with0.14mol/L NaCl solution to decrease the influence of RNP on extraction of DNP.

Extract the underlayer for one hour with twice volume cold NaClsolution of 1mol/L to separate DNP.

(2) Extraction of nucleic acid

① Extraction of RNA: Add equal volume of chloroform and 1/40 volumeof isoamylol to the extracting solution of 0.14mol/L NaCl (contain RNP), putinto centrifugal tube with stopper and shake it for 30 minutes. The extractingsolution is ivory suspension, centrifuge it for 15 minutes with 3000rpm. Thereare three layers in the centrifugal tube. (fig.10-1)

figure 10-1

upper－solution containednucleic acid；middle－denatured proteins；lower－chloroform

Remove the supernatant with dropper, add 1.5~2 times volume of 95%cold ethanol and stir gently. Keep for 15 minutes at low temperature,centrifuge for 15 minutes with 3000rpm, abandon the supernatant to give granularsediment of RNA for identification.

② Extraction of DNA (it is necessary to freezing-centrifuge everytime): Centrifuge the homogenate suspension which extracted for one hour with1mol/L NaCl solution, abandon the sediment, pour the supernatant intocentrifugal tube with stopper, add equal volume of chloroform and 1/40 volumeof isoamylol and shake it for 30 minutes, centrifuge for 20 minutes. Add twicevolume of 95% cold ethanol, shake at the same time, draw out glass stick, thefibrous DNA will enlace the glass stick. Wait to identify.

(2) Identification

① Color reaction of RNA: Take two clean test tubes, add 1.5ml of RNAsolution (dissolve granular sediment of RNA into 4ml of 0.14mol/L NaCl solutionto give RNA solution) into one tube and 1.5ml of distilled water into theother. Add 3ml of orcinol reagent in each of the two tube, heat them for tenminutes in boiling water. The color of positive reaction changes yellow togreen.

② Color reaction of DNA: Take two clean test tubes, add 1.5ml of DNAsolution (dissolve fibrous sediment of DNA into 4ml of 1mol/L NaCl solution togive DNA solution) into one tube and 1.5ml of distilled water into the other.Add 3ml of diphenylamine reagent in each of the two tube, heat them for tenminutes in boiling water. The color of positive reaction changes ivory to blue.

Experimental Guidance

1. Previewrequire

(1) Know the principle of preparing nucleicacid. The generic principle of extracting nucleic acid is to fragmentize cellsby mechanical method or enzymatic method, then to precipitate protein with denaturant(eg phenol) or detergent (eg 衛(wèi)生資格考試網(wǎng)SDS), then precipitate nucleic acid with ethanol.

(2) Nucleic acid is composed of saccharum(ribose or deoxyribose), phosphate, nitrogenous basic group (purine or pyrimidine).We can use color reaction (orcinol reaction and diphenylamine reaction) if wewant to identify RNA and DNA rapidly and simply.

2. Attention

(1) Avoid superacid and superalkali whenextracting and purifying nucleic acid to keep nucleic acid intact withoutdenaturation and degradation. The whole process should be at low temperature(about 0℃) and add inhibitor to inhibit the nuclease if needed.

(2) Shake up completely after addingchloroform and isoamylol but not too acutely to avoid rupture of DNA.

(3) Learn to use centrifuge rightly. Payattention to make choice of the supernatant and sediment after centrifugation.

3. Discussion

(1) A certain extent depolymerization ofDNA will not affect the identification.

(2) The identification methods of RNA andDNA can also be used to quantification.

4. Advisementafter experiment

(1) What shall we pay attention to duringexacting nucleic acid?

(2) Describe the distribution, existingmanner and characters of nucleic acid in the cells in a nutshell.

實(shí)驗(yàn)六  動(dòng)物組織中核酸的提取與鑒定

一、目的要求

1. 學(xué)習(xí)和掌握用鹽溶法從動(dòng)物組織提取核酸的原理和操作技術(shù)。

2. 了解定性鑒定核酸的原理和方法。

二、實(shí)驗(yàn)原理

根據(jù)核糖核蛋白與脫氧核糖核蛋白在一定濃度的氯化鈉溶液中的溶解度不同進(jìn)行分離，然后用蛋白質(zhì)變性沉淀劑去除蛋白，使核酸釋放出來(lái)，再利用核酸不溶于乙醇的性質(zhì)將核酸析出，達(dá)到分離提純的目的。

在0.14mol/L的氯化鈉溶液中，RNA核蛋白溶解度大，而DNA核蛋白溶解度較��；相反，在1mol/L的氯化鈉溶液中，DNA核蛋白溶解度最大，而RNA核蛋白溶解度卻很小。核蛋白分離后可用蛋白變性沉淀劑(氯仿+異戊醇)、十二烷基硫酸鈉(SDS)和熱酚等)去除蛋白，釋放核酸。去除蛋白后的核酸溶液中加入1.5~2倍體積的95%乙醇，核酸便從溶液中析出。

動(dòng)物肝中含有核糖核酸酶(RNase) 和脫氧核糖核酸酶(DNase)，因此要保持低溫，要防止Mg²⁺、Fe²⁺及Co²⁺等激活離子。

三、實(shí)驗(yàn)材料

(一) 試劑

1.地衣酚(orcinol)試劑：稱取100mg地衣酚溶于100ml濃鹽酸中，再加入100mg FeCl₃·6H₂O，該溶液應(yīng)在使用前新鮮配制。

2.二苯胺試劑：稱取1g二苯胺(經(jīng)重結(jié)晶)溶入100ml冰醋酸中，再加入2.75ml濃H₂SO₄搖勻，冰箱內(nèi)保存?zhèn)溆谩?/p>

3. 0.14mol/L氯化鈉溶液。

4. 1mol/L氯化鈉溶液。

5. 95%乙醇。

6. 氯仿。  

7. 丙酮。

8. 異戊醇。

(二) 器材

1. 冷凍離心機(jī)(3000r.p.m)。

2. 冰浴。

3. 組織搗碎機(jī)或組織勻漿器。

4. 燒杯。

5. 量筒。

6. 試管。

7. 玻棒。  

8. 剪刀。

9. 三角燒瓶或帶塞離心管。

四、實(shí)驗(yàn)方法

(一) 制勻漿

將新鮮肝臟或冷凍肝臟稱重后用冷的0.14mol/L氯化鈉溶液(內(nèi)含0.01mol/L檸檬酸)洗去血水，用不銹鋼剪刀將肝臟剪成碎塊，放入組織搗碎機(jī)中加入2倍體積的0.14mol/LNaCl溶液(含0.01mol/L檸檬酸)制備勻漿。

將勻漿置離心機(jī)中離心20min(3000r.p.m)。上層是RNA核蛋白提取液，下層是細(xì)胞碎片及DNA核蛋白。上層液傾出留待抽提RNA(下層再用0.14mol/L NaCl溶液重復(fù)抽提兩次)，以減少RNA核蛋白對(duì)DNA核蛋白抽提的影響。

下層用二倍體積冷的1mol/L NaCl溶液抽提1h，待分離DNA核蛋白。

(二) 核酸的抽提

1. RNA的提�。�0.14mol/L 的NaCl抽提液(內(nèi)含RNA核蛋白)加入等體積的氯仿和1/40體積的異戊醇，置帶塞離心管中，振搖30min，此時(shí)提取液為乳白色混懸液，以3000r.p.m離心15min，離心物呈三層(圖9-1)。

圖 9-1

上層-含核酸的溶液；中層-變性蛋白；下層-氯仿

    用滴管吸出上層清液，在低溫下加入1.5～2倍體積的冷95%乙醇，并輕輕攪拌。低溫放置15min，3000r.p.m離心10min，棄去上清液，即得RNA顆粒狀沉淀，待定性。

2. DNA的抽提(每次需采用冷凍離心)：將抽提1h的1mol/L NaCl勻漿懸液以3000r.p.m離心20min，棄去沉淀，將其上清液倒入帶塞的離心管中，加入等體積的氯仿及1/40體積的異戊醇振搖30min，離心20min。將其上清液加入2倍體積的冷95%乙醇，邊加邊搖，抽出玻棒，纖維狀DNA就纏繞在玻棒上待定性。

(三) 定性試驗(yàn)

1. RNA的呈色反應(yīng)：取2支干凈試管，一管加入1.5ml RNA溶液(將RNA顆粒狀的沉淀溶于4ml 0.14mol/LNaCl溶液中即為RNA提取液)，另一管加入蒸餾水1.5ml，向兩管中加入3ml地衣酚試劑，于沸水中加熱10min，由黃色變成綠色為陽(yáng)性反應(yīng)。

2. DNA的呈色反應(yīng)：取2支干凈試管，一管加入1.5ml DNA溶液(將DNA纖維狀沉淀溶于4ml 1mol/LNaCl溶液中即為DNA提取液)，另一管加入蒸餾水1.5ml，向兩管中加入3ml二苯胺試劑，于沸水中加熱10min，由乳白色變成藍(lán)色為陽(yáng)性反應(yīng)。

實(shí) 驗(yàn) 指 導(dǎo)

一、預(yù)習(xí)要求

1. 了解核酸制備的原理。提取核酸的一般原則是用機(jī)械國(guó)家醫(yī)學(xué)考試網(wǎng)方法或酶學(xué)方法(溶菌酶)使細(xì)胞破碎，然后用蛋白質(zhì)變性劑(如苯酚)、去垢劑(如十二烷基硫酸鈉)等處理，使蛋白質(zhì)沉淀，最后用乙醇將核酸沉淀。

2. 核酸是由糖(核糖或脫氧核糖)、磷酸和含氮堿基(嘌呤或嘧啶)所組成的。要快速簡(jiǎn)單地鑒定出RNA和DNA，可采用呈色反應(yīng)(地衣酚反應(yīng)、二苯胺反應(yīng))。

二、注意事項(xiàng)

1. 在提取分離純化核酸時(shí)，為了保持核酸的完整性，防止核酸變性降解，操作時(shí)必須防止過(guò)酸或過(guò)堿。全部過(guò)程應(yīng)在低溫下(0℃左右)進(jìn)行，必要時(shí)還需加入抑制劑，以抑制核酸酶的作用。

2. 加氯仿、異戊醇后振搖要充分，但又不能太劇烈，以防DNA斷裂。

3. 要學(xué)會(huì)正確使用離心機(jī)，離心后，注意上清液和沉淀的取舍。

三、結(jié)果討論

1. 所得的DNA若有一定程度解聚，不會(huì)影響鑒定。

2. RNA、DNA鑒定方法可用于定量。

四、實(shí)驗(yàn)后思考

1. 在提取核酸過(guò)程中，要注意些什么問(wèn)題？

2. 簡(jiǎn)述核酸在細(xì)胞內(nèi)的分布、存在方式及其特性。