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.
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 卫生资格考试网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.
实验六 动物组织中核酸的提取与鉴定
一、目的要求
1. 学习和掌握用盐溶法从动物组织提取核酸的原理和操作技术。
2. 了解定性鉴定核酸的原理和方法。
二、实验原理
根据核糖核蛋白与脱氧核糖核蛋白在一定浓度的氯化钠溶液中的溶解度不同进行分离，然后用蛋白质变性沉淀剂去除蛋白，使核酸释放出来，再利用核酸不溶于乙醇的性质将核酸析出，达到分离提纯的目的。
在0.14mol/L的氯化钠溶液中，RNA核蛋白溶解度大，而DNA核蛋白溶解度较小；相反，在1mol/L的氯化钠溶液中，DNA核蛋白溶解度最大，而RNA核蛋白溶解度却很小。核蛋白分离后可用蛋白变性沉淀剂(氯仿+异戊醇)、十二烷基硫酸钠(SDS)和热酚等)去除蛋白，释放核酸。去除蛋白后的核酸溶液中加入1.5~2倍体积的95%乙醇，核酸便从溶液中析出。
动物肝中含有核糖核酸酶(RNase) 和脱氧核糖核酸酶(DNase)，因此要保持低温，要防止Mg2+、Fe2+及Co2+等激活离子。
三、实验材料
(一) 试剂
1.地衣酚(orcinol)试剂：称取100mg地衣酚溶于100ml浓盐酸中，再加入100mg FeCl3·6H2O，该溶液应在使用前新鲜配制。
2.二苯胺试剂：称取1g二苯胺(经重结晶)溶入100ml冰醋酸中，再加入2.75ml浓H2SO4摇匀，冰箱内保存备用。
3. 0.14mol/L氯化钠溶液。
4. 1mol/L氯化钠溶液。
5. 95%乙醇。
6. 氯仿。
7. 丙酮。
8. 异戊醇。
(二) 器材
1. 冷冻离心机(3000r.p.m)。
2. 冰浴。
3. 组织捣碎机或组织匀浆器。
4. 烧杯。
5. 量筒。
6. 试管。
7. 玻棒。
8. 剪刀。
9. 三角烧瓶或带塞离心管。
四、实验方法
(一) 制匀浆
将新鲜肝脏或冷冻肝脏称重后用冷的0.14mol/L氯化钠溶液(内含0.01mol/L柠檬酸)洗去血水，用不锈钢剪刀将肝脏剪成碎块，放入组织捣碎机中加入2倍体积的0.14mol/LNaCl溶液(含0.01mol/L柠檬酸)制备匀浆。
将匀浆置离心机中离心20min(3000r.p.m)。上层是RNA核蛋白提取液，下层是细胞碎片及DNA核蛋白。上层液倾出留待抽提RNA(下层再用0.14mol/L NaCl溶液重复抽提两次)，以减少RNA核蛋白对DNA核蛋白抽提的影响。
下层用二倍体积冷的1mol/L NaCl溶液抽提1h，待分离DNA核蛋白。
(二) 核酸的抽提
1. RNA的提取：0.14mol/L 的NaCl抽提液(内含RNA核蛋白)加入等体积的氯仿和1/40体积的异戊醇，置带塞离心管中，振摇30min，此时提取液为乳白色混悬液，以3000r.p.m离心15min，离心物呈三层(图9-1)。
图 9-1
上层-含核酸的溶液；中层-变性蛋白；下层-氯仿
用滴管吸出上层清液，在低温下加入1.5～2倍体积的冷95%乙醇，并轻轻搅拌。低温放置15min，3000r.p.m离心10min，弃去上清液，即得RNA颗粒状沉淀，待定性。
2. DNA的抽提(每次需采用冷冻离心)：将抽提1h的1mol/L NaCl匀浆悬液以3000r.p.m离心20min，弃去沉淀，将其上清液倒入带塞的离心管中，加入等体积的氯仿及1/40体积的异戊醇振摇30min，离心20min。将其上清液加入2倍体积的冷95%乙醇，边加边摇，抽出玻棒，纤维状DNA就缠绕在玻棒上待定性。
(三) 定性试验
1. RNA的呈色反应：取2支干净试管，一管加入1.5ml RNA溶液(将RNA颗粒状的沉淀溶于4ml 0.14mol/LNaCl溶液中即为RNA提取液)，另一管加入蒸馏水1.5ml，向两管中加入3ml地衣酚试剂，于沸水中加热10min，由黄色变成绿色为阳性反应。
2. DNA的呈色反应：取2支干净试管，一管加入1.5ml DNA溶液(将DNA纤维状沉淀溶于4ml 1mol/LNaCl溶液中即为DNA提取液)，另一管加入蒸馏水1.5ml，向两管中加入3ml二苯胺试剂，于沸水中加热10min，由乳白色变成蓝色为阳性反应。
实 验 指 导
一、预习要求
1. 了解核酸制备的原理。提取核酸的一般原则是用机械国家医学考试网方法或酶学方法(溶菌酶)使细胞破碎，然后用蛋白质变性剂(如苯酚)、去垢剂(如十二烷基硫酸钠)等处理，使蛋白质沉淀，最后用乙醇将核酸沉淀。
2. 核酸是由糖(核糖或脱氧核糖)、磷酸和含氮碱基(嘌呤或嘧啶)所组成的。要快速简单地鉴定出RNA和DNA，可采用呈色反应(地衣酚反应、二苯胺反应)。
二、注意事项
1. 在提取分离纯化核酸时，为了保持核酸的完整性，防止核酸变性降解，操作时必须防止过酸或过碱。全部过程应在低温下(0℃左右)进行，必要时还需加入抑制剂，以抑制核酸酶的作用。
2. 加氯仿、异戊醇后振摇要充分，但又不能太剧烈，以防DNA断裂。
3. 要学会正确使用离心机，离心后，注意上清液和沉淀的取舍。
三、结果讨论
1. 所得的DNA若有一定程度解聚，不会影响鉴定。
2. RNA、DNA鉴定方法可用于定量。
四、实验后思考
1. 在提取核酸过程中，要注意些什么问题？
2. 简述核酸在细胞内的分布、存在方式及其特性。
