生物技术综合实验.ppt

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1、生物技术综合实验Comprehensive experiments of biotechnology 主要内容 Contents：一、课程简介 核酸的分离与纯化 Isolation and Purification of Nucleic Acid二、电泳技术 Agarose Gel Electrophoresis and SDS-PAGE 三、聚合酶链式反应技术 Polymerase Chain Reaction四、DNA序列测定 DNA Sequencing五、分子杂交技术 Molecular Hybridization Southern,Northern and Western Blot六

2、、基因文库和cDNA文库的构建 Construction of cDNA Library and DNA Library 七、外源基因的克隆与表达 Heterogenic Gene Cloning and Expression八、蛋白质的分离、纯化技术 Isolation and Purification of Protein课程简介 现代生物技术综合性实验是以分子生物学为基础的基因克隆重组技术和生物化学的蛋白质分离、纯化技术为核心的教学，它是现代生物技术的核心。主要内容：包括实验理论和实验技术 实验技术生物技术综合实验，它是集目的基因的制备、克隆、表达和蛋白质的分离纯化及其活性测定等实验方法

3、和技术为一体的一门综合性实验课。目的：通过本门课程的学习，使学生掌握现代生物工程的上、下游实验技术，对以基因克隆重组技术为主线的生物实验技术有一个较全面的了解。教学要求 u通过教学，要求学生掌握分子生物学与基因工程的通过教学，要求学生掌握分子生物学与基因工程的基本理论，巩固所学的理论知识；基本理论，巩固所学的理论知识；u使学生了解科研工作的基本思路，学会如何设计实使学生了解科研工作的基本思路，学会如何设计实验，如何分析实验，培养分析问题和解决问题的能验，如何分析实验，培养分析问题和解决问题的能力；力；u培养和训练学生的基本实验技能，培养学生的独立培养和训练学生的基本实验技能，培养学生的独立工作

4、能力和创造能力。工作能力和创造能力。u掌握基因重组的基本过程，如：核酸掌握基因重组的基本过程，如：核酸DNADNA、RNARNA和质粒和质粒DNADNA提取、酶切、连接、转化及重组子的提取、酶切、连接、转化及重组子的酶切鉴定等技术。酶切鉴定等技术。u掌握外源基因的表达技术和蛋白质电泳分析技术，掌握外源基因的表达技术和蛋白质电泳分析技术，蛋白质提取、分离和纯化技术。蛋白质提取、分离和纯化技术。核酸的分离与纯化Isolation and purification of nucleic acids 不论是基因工程还是蛋白质工程，核酸分子是这些技术应用所涉及的主要对象，所以核酸的分离与提取是生化与分子

5、生物学研究中非常重要的基本技术。核酸样品的质量将直接关系到实验的成败。核酸的种类：真核生物的染色体DNA为双链线性分子；真核细胞器DNA为双链环状分子；原核生物的基因组DNA、质粒DNA为双链环状分子；RNA分子在大多数生物体内均是单链线性分子；不同类型的RNA分子可具有不同的结构特点，如真核mRNA分子多数在3端带有ploy(A)结构;tRNA 的三叶草结构。病毒的DNA、RNA，其存在形式多种多样，有双链环状、单链环状、双链线状和单链线状等。一、核酸分离提取的原则 1.分离纯化核酸总的原则 2.应保证核酸一级结构的完整性；3.排除其它分子的污染，保证较高纯度。2.核酸的分离、纯化应达到以下

6、三点要求：排除其它核酸分子的污染，如提取DNA分子时，应去除RNA分子，反之亦然；其它生物大分子如蛋白质、多糖和脂类分子的污染应降低到最低程度；核酸样品中不应存在对酶有抑制作用的有机溶剂和过高浓度的金属离子。3.为了保征分离核酸的完整性和纯度，在实验过程中，应注意以下事宜：尽量简化操作步骤，缩短提取过程，以减少各种有害因素对核酸的破坏；减少化学因素对核酸的降解，为避免过酸、过碱对核酸链中磷酸二酯键的破坏，操作多在pH4-10条件下进行；减少物理因素对核酸的破坏，物理破坏因素主要是机械剪切力，其次是高温。防止核酸的生物降解，细胞内或外界的各种核酸酶（DNA酶、RNA酶）酶解核酸链中的磷酸二酯键，

7、直接破坏核酸的一级结构。其中DNA酶，需要金属二价离子Mg2+，Ca2+的激活，使用金属二价离子螯合剂乙二胺四乙酸(EDTA)、柠檬酸盐，基本上可以抑制DNA酶的活性。而RNA酶，不但分布广泛、极易污染样品，而且耐高温、耐酸、耐碱，不易失活，所以生物降解是RNA提取过程中的主要危害因素。nn 掌握真核生物基因组DNA的提取的基本原理。nn 提取高质量基因组DNA的主要用途：PCR扩增的模板 用于构建基因组文库 用于Southern blot 杂交分析二、真核细胞染色体DNA的制备 真核细胞染色体DNA的制备过程 1.真核细胞的破碎（1）物理方式:超声波法、匀浆法、液氮破碎法、Al2O3粉研磨法

8、等。这些物理操作均可导致DNA链的断裂。（2）为了获得大分子量的DNA，一般采用蛋白酶K和去污剂温和处理法。2.去除蛋白质 常用酚、氯仿抽提。反复的抽提操作对DNA链的机械剪切机会较多，所以有人使用高浓度甲酰胺解聚核蛋白联合透析的方法，可以获得200kb以上的DNA片段，适用于粘粒(cosmid)构建基因组文库。根据不同的实验要求，可选择不同的实验方法制备真核细胞染色体DNA。3.DNA沉淀：一般采用2倍体积乙醇沉淀 或用异丙醇（0.6）4.DNA溶解：一般采用TE方法一：酚抽提法（见实验指导p20）DNA Extraction n n 从外周血提取基因组DNA的步骤 PROCEDURE OF

9、 DNA EXTRACTION FROM BLOOD.doc三、RNA的分离与纯化-真核细胞RNA的制备 从细胞中分离RNA的纯度与完整性对于许多分子生物学实验至关重要。如Northern blot及cDNA合成及体外翻译等实验的成败，在很大程度上决定于RNA的质量。RNA主要由以下几类分子组成：rRNA(占RNA总量的8085)、tRNA和核内小分子RNA(占10-15)、mRNA(占15)。rRNA在总RNA分子中含量最丰富，由28S、18S、5S及4S几类组成，它们之间同源性大，分子量变化不大，所以可根据它们的密度和分子大小，通过密度梯度离心，凝胶电泳或离子交换层析进行分离。mRNA分子

10、种类繁多，分子量大小不均一，在细胞中含量少，绝大多数mRNA分子(除血红蛋白及有些组蛋白mRNA以外)，均在3端存在20-250个多聚腺核苷酸poly(A)。利用此特征，可很方便地从总RNA中，用寡聚(dT)亲和层析柱分离mRNA。mRNA分子群体编码细胞内所有的多肽和蛋白质，而mRNA是分子生物学的主要研究对象之一。鉴定所提取总RNA分子的质量，可以通过琼脂糖电泳后观察是否有明显得28S、18S条带来判断。Fig.1 Total RNA isolated from Trichoderma reesei induced by different inducers图1.木霉总RNA的分离1.微晶

11、纤维素 2.天然稻草粉RNA 分离的关键因素是尽量减少RNA酶的污染！如何创造一个无RNase的环境？极力避免外源RNase的污染：主要来源于操作者的手、实验的器皿和试剂尽力抑制内源性RNase的活力：主要来源于样品中的组织细胞。(1)操作者的手直接触摸之处，毫无疑问会留下RNase，说话带出的唾液也富含RNase。故整个操作过程中，应戴口罩和手套。(2)空气中飞尘携带的细菌、霉菌等微生物，也是污染外源RNase的一条途径，所以操作过程应在比较洁净的环境中进行。怎样去除外源性RNase的污染？(3)玻璃器经过常规洗净后，应用0.1焦碳酸二乙酯(diethyl pyrocarbonate，DEP

12、C)浸泡处理(37，2小时)，再用双蒸灭菌水漂洗几次。高压消毒去除DEPC，然后200烘烤4小时以上或180烘烤过夜。(4)塑料器材最好使用灭菌的一次性塑料用品，Eppendorf管、微量加样吸头最好是新的，使用前进行高压蒸汽消毒。(5)所有溶液应加DEPC至0.1，室温处理过夜，或室温下磁力搅拌20分钟，然后高压蒸汽灭菌处理（或加热至701小时或60过夜，以去除所有残留的DEPC）。(6)所用的化学试剂应为新包装，称量时使用干烤处理的称量勺。所有操作均应在冰浴中进行，低温条件可减低RNA酶的活性。要尽可能早地去除细胞内蛋白，加入RNase抑制剂，力争在提取的起始阶段对RNase活力进行有效地

13、抑制。如何抑制内源性RNase的活性？RNase抑制物：（1）低特异性RNase抑制物：不同类型的低特异性RNase抑制物均可不同程度地抑制Raise的活性，其中包括DEPC 皂土(bentonite):Al2O34SiO2H2O复合硅酸盐(Macaloid)肝素氧钒基核苷复合物多胺（2）去除蛋白质的物质：蛋白质变性剂：酚、氯仿；尿素蛋白酶K去污剂：SDS、十二烷酰肌氨酸钠(sarkosyl)、脱氧胆酸钠(DOC)是阴离子去污剂解偶剂(胍类)：盐酸胍、异硫氰酸胍是一类强力的蛋白质变性剂，可溶解蛋白质，并使蛋白质二级结构消失，细胞结构降解，核蛋白迅速与核酸分离，所以又称解偶剂。（3）RNase的

14、特异抑制剂 RNase阻抑蛋白(RNasin)：这是一类从大鼠肝或人胎盘中提取出来的酸性糖蛋白质。RNasin能与RNase非共价结合从而抑制它们的活力，RNasin是RNA酶的一种非竞争性抑制剂。抑制效果较好，被广泛用于体外翻译体系、mRNA反转录合成cDNA及体外转录实验。RNA的提取方法（见有关实验指南）Acid-guanidinium thiocyanate-phenol-chloroform RNA purification(after Chomczynski after Chomczynski and Sacchi,1987 and Sacchi,1987)n n N.B:Care

15、 must be taken when handling solutions containing high concentrations of guanidine salts due to its chaotropic nature.As with other procedures involving RNA,gloves should be worn at all times to avoid contamination of samples with ribonucleases.This method describes preparation from small quantities

16、(50mg)of tissue.Appropriate scaling of the volumes involved can be performed to accomodate larger quantities.n n You will need:You will need:n n Guanidine isothiocyanate(Sigma)Guanidine isothiocyanate(Sigma)1M sodium citrate,pH 7(DEPC-treated,autoclaved)1M sodium citrate,pH 7(DEPC-treated,autoclaved

17、)Sarcosyl(Na-lauryl sarcosine,Sigma)Sarcosyl(Na-lauryl sarcosine,Sigma)b-mercaptoethanol(Sigma)b-mercaptoethanol(Sigma)2M sodium acetate,pH4(DEPC-treated,autoclaved)2M sodium acetate,pH4(DEPC-treated,autoclaved)3M sodium acetate,100mM magnesium acetate,pH 5.2 3M sodium acetate,100mM magnesium acetat

18、e,pH 5.2Absolute ethanol Absolute ethanolPropan-2-ol(isopropanol)Propan-2-ol(isopropanol)70%ethanol(made with DEPC-treated,autoclaved water)70%ethanol(made with DEPC-treated,autoclaved water)0.5%SDS(made with sterile,DEPC-treated water)0.5%SDS(made with sterile,DEPC-treated water)Solution D-4M guani

19、dine isothiocyanate,25mM sodium citrate,Solution D-4M guanidine isothiocyanate,25mM sodium citrate,pH7,0.5%sarcosyl,100mM b-mercaptoethanol.pH7,0.5%sarcosyl,100mM b-mercaptoethanol.n n Solution D can be made and stored at 4C without b-mercaptoethanol Solution D can be made and stored at 4C without b

20、-mercaptoethanol for several months.b-mercaptoethanol should be added to 100mM for several months.b-mercaptoethanol should be added to 100mM immediately prior to use.b-mercaptoethanol is used to prevent the immediately prior to use.b-mercaptoethanol is used to prevent the reformation of the intra-mo

21、lecular disulphide bridges,one of the reformation of the intra-molecular disulphide bridges,one of the reasons for the extreme stability of ribonucleases.reasons for the extreme stability of ribonucleases.n n 1)Tissue is homogenized as rapidly as possible,at 4C,in solution D(500ul per 1)Tissue is ho

22、mogenized as rapidly as possible,at 4C,in solution D(500ul per 50mg tissue)with an eppendorf pestle homogenizer until a smooth,lysed,50mg tissue)with an eppendorf pestle homogenizer until a smooth,lysed,homogenous suspension is obtained.homogenous suspension is obtained.n n 2)Add 50ul 2M sodium acet

23、ate,pH4.0 and mix vigorously.2)Add 50ul 2M sodium acetate,pH4.0 and mix vigorously.n n 3)Add 500ul phenol and mix vigorously.3)Add 500ul phenol and mix vigorously.n n 4)Add 100ul chloroform,mix vigorously and incubate on ice for 15 minutes.4)Add 100ul chloroform,mix vigorously and incubate on ice fo

24、r 15 minutes.n n 5)Centrifuge mixture at 10,000g for 10 minutes in a microfuge at 4C.5)Centrifuge mixture at 10,000g for 10 minutes in a microfuge at 4C.n n 6)Remove upper,aqueous phase to a clean,sterile,DEPC-treated eppendorf 6)Remove upper,aqueous phase to a clean,sterile,DEPC-treated eppendorf t

25、ube.After centrifugation,RNA is present in the aqueous phase while,due to tube.After centrifugation,RNA is present in the aqueous phase while,due to protonation at the acidic pH used,genomic DNA is partitioned into the phenol protonation at the acidic pH used,genomic DNA is partitioned into the phen

26、ol phase.phase.n n 7)Extract the upper aqueous layer with an equal volume phenol/chloroform and 7)Extract the upper aqueous layer with an equal volume phenol/chloroform and centrifuge as before.Repeat the extractions until no interface material is seen.centrifuge as before.Repeat the extractions unt

27、il no interface material is seen.n n 8)Precipitate the aqueous phase by the addition of an equal volume(500ul)of 8)Precipitate the aqueous phase by the addition of an equal volume(500ul)of propan-2-ol.Incubate at-20C for 20 minutes.propan-2-ol.Incubate at-20C for 20 minutes.n n 9)Pellet RNA by centr

28、ifugation at maximum speed in a microfuge for 10 9)Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes.minutes.n n 10)Wash the RNA once in 70%ethanol and vacuum dry.10)Wash the RNA once in 70%ethanol and vacuum dry.n n 11)Re-dissolve in 200ul 0.5%SDS at 65C.11)Re-dissolve in

29、200ul 0.5%SDS at 65C.n n 12)Extract with an equal volume(200ul)of phenol/chloroform as above.12)Extract with an equal volume(200ul)of phenol/chloroform as above.Repeat until no interface material is visible.Repeat until no interface material is visible.n n 13)Precipitate pure RNA by the addition of

30、20ul 3M sodium acetate,100mM 13)Precipitate pure RNA by the addition of 20ul 3M sodium acetate,100mM acetate,pH 5.2 and 500ul absolute ethanol.Incubate at-20C for 20 minutes.acetate,pH 5.2 and 500ul absolute ethanol.Incubate at-20C for 20 minutes.n n 14)Pellet RNA by centrifugation at maximum speed

31、in a microfuge for 10 14)Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes.minutes.n n 15)Wash the RNA once in 70%ethanol and vacuum dry.15)Wash the RNA once in 70%ethanol and vacuum dry.n n 16)Dissolve RNA in appropriate buffer i.e.DEPC-treated,sterile TE,pH 8 or 16)Dissol

32、ve RNA in appropriate buffer i.e.DEPC-treated,sterile TE,pH 8 or 0.5%SDS if no enzymic manipulation of the RNA is needed.SDS is an 0.5%SDS if no enzymic manipulation of the RNA is needed.SDS is an inhibitor of ribonucleases.inhibitor of ribonucleases.n n RNA quality can be assessed by electrophoresi

33、s under denaturing conditions RNA quality can be assessed by electrophoresis under denaturing conditions using agarose/formaldehyde gels and the MOPS buffer system.using agarose/formaldehyde gels and the MOPS buffer system.质粒DNA的提取DNA Plasmid Miniprep Protocol n n1.Pick single colony and inoculate 5

34、 ml of 1.Pick single colony and inoculate 5 ml of LB brothLB broth containing 200 g/l ampicillin or 1mg/5ml.Optional:Use containing 200 g/l ampicillin or 1mg/5ml.Optional:Use a 15ml conical tube with a loosened cap and a piece a 15ml conical tube with a loosened cap and a piece of tape to hold it in

35、 place.Shake at 250 RPM 37of tape to hold it in place.Shake at 250 RPM 37ooC C overnight.overnight.n n2.Centrifuge 1.5mL cells in 1.5 mL Eppendorf tube at 2.Centrifuge 1.5mL cells in 1.5 mL Eppendorf tube at top speed for 1 minute.Aspirate supernatant.top speed for 1 minute.Aspirate supernatant.n n3

36、.Resuspend cell pellet in 100 ul of 3.Resuspend cell pellet in 100 ul of GTE bufferGTE buffer(50(50 mM Glucose,25 mM Tris-Cl,10 mM EDTA,pH 8).mM Glucose,25 mM Tris-Cl,10 mM EDTA,pH 8).Vortex gently if necessary.Vortex gently if necessary.n n4.Add 200 ul of 4.Add 200 ul of NaOH/SDS lysis solutionNaOH

37、/SDS lysis solution(0.2 M(0.2 M NaOH,1%SDS).Invert tube 6-8 times.NaOH,1%SDS).Invert tube 6-8 times.n n5.IMMEDIATELY add 150 ul of 5.IMMEDIATELY add 150 ul of 5 M potassium 5 M potassium acetate solution(pH 4.8)acetate solution(pH 4.8).This solution neutralizes.This solution neutralizes NaOH in the

38、previous lysis step while precipitating the NaOH in the previous lysis step while precipitating the genomic DNA and SDS in an insoluble white,rubbery genomic DNA and SDS in an insoluble white,rubbery precipitate.Spin at top speed 1 min.precipitate.Spin at top speed 1 min.n n6.Transfer supernatant to

39、 new tube,being careful not 6.Transfer supernatant to new tube,being careful not to pick up any white flakes.Precipitate the nucleic to pick up any white flakes.Precipitate the nucleic acids with 0.5mL of isopropanol on ice for 10 minutes acids with 0.5mL of isopropanol on ice for 10 minutes and cen

40、trifuge at top speed for 1 minute.and centrifuge at top speed for 1 minute.n n7.Aspirate off all the isopropanol supernatant.7.Aspirate off all the isopropanol supernatant.Dissolve the pellet in 0.4 ml of Dissolve the pellet in 0.4 ml of TE bufferTE buffer(10 mM Tris-(10 mM Tris-Cl,1 mM EDTA,pH 7.5)

41、.Add 10ul of RNAse A Cl,1 mM EDTA,pH 7.5).Add 10ul of RNAse A solution(20 mg/ml stock stored at-20 C),vortex and solution(20 mg/ml stock stored at-20 C),vortex and incubate at 37 C for 20 to 30 minutes to digest incubate at 37 C for 20 to 30 minutes to digest remaining RNA.remaining RNA.n n8.Extract

42、 proteins from the plasmid DNA using PCIA 8.Extract proteins from the plasmid DNA using PCIA(phenol/chloroform/isoamyl alcohol)by adding about(phenol/chloroform/isoamyl alcohol)by adding about 0.3 ml.Vortex vigorously for 30 seconds.Centrifuge at 0.3 ml.Vortex vigorously for 30 seconds.Centrifuge at

43、 full speed for 5 minutes at room temperature.Note full speed for 5 minutes at room temperature.Note organic PCIA layer will be at the bottom of the tube.organic PCIA layer will be at the bottom of the tube.n n9.Remove upper aqueous layer containing the 9.Remove upper aqueous layer containing the pl

44、asmid DNA carefully avoiding the white precipitated plasmid DNA carefully avoiding the white precipitated protein layer above the PCIA layer,transferring to a protein layer above the PCIA layer,transferring to a clean 1.5 ml epindorf tube.clean 1.5 ml epindorf tube.n n10.Add 100 10.Add 100 mml of 7.

45、5 M ammonium acetate solution l of 7.5 M ammonium acetate solution and 1 ml of absolute ethanol to precipitate the plasmid and 1 ml of absolute ethanol to precipitate the plasmid DNA on ice for 10 minutes.Centrifuge at full speed for DNA on ice for 10 minutes.Centrifuge at full speed for 5 minutes a

46、t room temperature.5 minutes at room temperature.n n11.Aspirate off ethanol solution and resuspend or 11.Aspirate off ethanol solution and resuspend or dissolve DNA pellet in 50ul of DNA.Dissolve 5uL in dissolve DNA pellet in 50ul of DNA.Dissolve 5uL in 995ul of water,and spec(blank spectrophotomete

47、r to 995ul of water,and spec(blank spectrophotometer to water).The absorbance at 260 nm multiplied by ten is water).The absorbance at 260 nm multiplied by ten is the concentration of the DNA in units of mg/ml for a 1 the concentration of the DNA in units of mg/ml for a 1 cm pathlength cuvette(i.e.50 cm pathlength cuvette(i.e.50 mmg/ml/OD 260nm).g/ml/OD 260nm).Fig.2.egl4 gene was amplified by RT-PCR图2.RT-PCR扩增基因1.内切葡萄糖苷酶基因2.DNA 标准分子量