生物技术综合实验生物技术综合实验Comprehensive experiments of biotechnology 主要内容主要内容 ContentsContents：一、课程简介一、课程简介 核酸的分离与纯化核酸的分离与纯化 Isolation and Purification of Nucleic Acid二、电泳技术二、电泳技术 Agarose Gel Electrophoresis and SDS-PAGE 三、聚合酶链式反应技术三、聚合酶链式反应技术 Polymerase Chain Reaction四、四、DNADNA序列测定序列测定 DNA Sequencing五、分子杂交技术五、分子杂交技术 Molecular Hybridization Southern, Northern and Western Blot六、基因文库和六、基因文库和cDNAcDNA文库的构建文库的构建 Construction of cDNA Library and DNA Library 七、外源基因的克隆与表达七、外源基因的克隆与表达 Heterogenic Gene Cloning and Expression八、蛋白质的分离、纯化技术八、蛋白质的分离、纯化技术 Isolation and Purification of Protein课程简介课程简介 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基因克隆重组技术重组技术和生物化学的蛋白质分离、纯化技术蛋白质分离、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和技术为一体的一门综合性实验课。 目的目的：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；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；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能力和创造能力。工作能力和创造能力。工作能力和创造能力。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DNADNADNADNA、RNARNARNARNA和质和质和质和质粒粒粒粒DNADNADNADNA提取、酶切、连接、转化及重组子的酶切鉴提取、酶切、连接、转化及重组子的酶切鉴提取、酶切、连接、转化及重组子的酶切鉴提取、酶切、连接、转化及重组子的酶切鉴定等技术。定等技术。定等技术。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，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。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生物学研究中非酸的分离与提取是生化与分子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。系到实验的成败。 核酸的种类：核酸的种类：真核生物的染色体真核生物的染色体DNADNA为双链线性分子；真核细胞为双链线性分子；真核细胞器器DNADNA为双链环状分子；为双链环状分子；原核生物的基因组原核生物的基因组DNADNA、质粒质粒DNADNA为双链环状分子；为双链环状分子；RNARNA分子在大多数生物体内均是单链线性分子；不分子在大多数生物体内均是单链线性分子；不同类型的同类型的RNARNA分子可具有不同的结构特点，如真核分子可具有不同的结构特点，如真核mRNAmRNA分子多数在分子多数在33端带有端带有ploy(A)ploy(A)结构结构; ;tRNAtRNA 的三的三叶草结构。叶草结构。病毒的病毒的DNADNA、RNARNA，其存在形式多种多样，有双链环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。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 1.1.分离纯化核酸总的原则分离纯化核酸总的原则 2.2.应保证核酸一级结构的完整性；应保证核酸一级结构的完整性；3.3.排除其它分子的污染，保证较高纯度。排除其它分子的污染，保证较高纯度。2.核酸的分离、纯化应达到以下三点要求：核酸的分离、纯化应达到以下三点要求：排排除除其其它它核核酸酸分分子子的的污污染染，如如提提取取DNADNA分分子子时，应去除时，应去除RNARNA分子，反之亦然；分子，反之亦然；其其它它生生物物大大分分子子如如蛋蛋白白质质、多多糖糖和和脂脂类类分分子的污染应降低到最低程度；子的污染应降低到最低程度；核核酸酸样样品品中中不不应应存存在在对对酶酶有有抑抑制制作作用用的的有有机溶剂和过高浓度的金属离子。机溶剂和过高浓度的金属离子。3.为为了了保保征征分分离离核核酸酸的的完完整整性性和和纯纯度度，在在实实验验过程中，应注意以下事宜：过程中，应注意以下事宜：尽尽量量简简化化操操作作步步骤骤，缩缩短短提提取取过过程程，以以减减少少各种有害因素对核酸的破坏；各种有害因素对核酸的破坏；减减少少化化学学因因素素对对核核酸酸的的降降解解，为为避避免免过过酸酸、过过碱碱对对核核酸酸链链中中磷磷酸酸二二酯酯键键的的破破坏坏，操操作作多多在在pH4-10pH4-10条件下进行；条件下进行；减减少少物物理理因因素素对对核核酸酸的的破破坏坏，物物理理破破坏坏因因素素主要是机械剪切力，其次是高温。主要是机械剪切力，其次是高温。 防防止止核核酸酸的的生生物物降降解解，细细胞胞内内或或外外界界的的各各种种核核酸酸酶酶（ DNADNA酶酶、 RNARNA酶酶）酶酶解解核核酸酸链链中中的的磷磷酸二酯键，直接破坏核酸的一级结构。酸二酯键，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。 n n掌握真核生物基因组DNA的提取的基本原理。n n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、真核细胞染色体二、真核细胞染色体DNADNA的制备的制备 真核细胞染色体真核细胞染色体DNADNA的制备过程的制备过程 1. 1. 真核细胞的破碎真核细胞的破碎 （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。 沉淀：一般采用2倍体积乙醇沉淀 或用异丙醇（）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从外周血提取基因组DNA的步骤的步骤 PROCEDURE OF DNA EXTRACTION FROM 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. 微晶纤维素 2. 天然稻草粉RNA RNA 分离的关键因素是尽量减少分离的关键因素是尽量减少RNARNA酶的污染！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，应用焦碳酸二乙酯(diethyl pyrocarbonate，DEPC)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，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。 RNARNA的提取方法（的提取方法（见有关有关实验指南）指南） Acid-guanidinium thiocyanate-phenol-chloroform RNA purification (after Chomczynski after Chomczynski and Sacchi, 1987and Sacchi, 1987)n nN.B: Care 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 ( 50mg) of tissue. Appropriate scaling of the volumes involved can be performed to accomodate larger quantities. n nYou will need: You will need: n nGuanidine isothiocyanate (Sigma)Guanidine isothiocyanate (Sigma)1M sodium citrate, pH 7 (DEPC-treated, autoclaved)1M sodium citrate, pH 7 (DEPC-treated, autoclaved)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 5.23M sodium acetate, 100mM magnesium acetate, pH 5.2Absolute ethanolAbsolute 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 guanidine 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 nSolution D can be made and stored at 4C without b-mercaptoethanol Solution D can be made and stored at 4C without b-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-molecular 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 n1) Tissue is homogenized as rapidly as possible, at 4C, in solution D (500ul per 1) Tissue is homogenized 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 n2) Add 50ul 2M sodium acetate, pH4.0 and mix vigorously. 2) Add 50ul 2M sodium acetate, pH4.0 and mix vigorously. n n3) Add 500ul phenol and mix vigorously. 3) Add 500ul phenol and mix vigorously. n n4) Add 100ul chloroform, mix vigorously and incubate on ice for 15 minutes. 4) Add 100ul chloroform, mix vigorously and incubate on ice for 15 minutes. n n5) 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 n6) Remove upper, aqueous phase to a clean, sterile, DEPC-treated eppendorf 6) Remove upper, aqueous phase to a clean, sterile, DEPC-treated eppendorf tube. 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 phenol phase. phase. n n7) 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 until no interface material is seen. n n8) 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 n9) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 9) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes. minutes. n n10) Wash the RNA once in 70% ethanol and vacuum dry. 10) Wash the RNA once in 70% ethanol and vacuum dry. n n11) Re-dissolve in 200ul 0.5% SDS at 65C. 11) Re-dissolve in 200ul 0.5% SDS at 65C. n n12) 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 n13) Precipitate pure RNA by the addition of 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 n14) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 14) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes. minutes. n n15) Wash the RNA once in 70% ethanol and vacuum dry. 15) Wash the RNA once in 70% ethanol and vacuum dry. n n16) Dissolve RNA in appropriate buffer i.e. DEPC-treated, sterile TE, pH 8 or 16) Dissolve 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 nRNA quality can be assessed by electrophoresis 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 ml of 1. Pick single colony and inoculate 5 ml of LB LB brothbroth containing 200 g/l ampicillin or 1mg/5ml. containing 200 g/l ampicillin or 1mg/5ml. Optional: Use a 15ml conical tube with a Optional: Use a 15ml conical tube with a loosened cap and a piece of tape to hold it in loosened cap and a piece of tape to hold it in place. Shake at 250 RPM 37place. Shake at 250 RPM 37o oC overnight. C overnight. n n2. Centrifuge 1.5mL cells in 1.5 mL Eppendorf 2. Centrifuge 1.5mL cells in 1.5 mL Eppendorf tube at top speed for 1 minute. Aspirate tube at top speed for 1 minute. Aspirate supernatant. supernatant. n n3. Resuspend cell pellet in 100 ul of 3. Resuspend cell pellet in 100 ul of GTE bufferGTE buffer (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 8). Vortex gently if necessary. 8). Vortex gently if necessary. n n4. Add 200 ul of 4. Add 200 ul of NaOH/SDS lysis solutionNaOH/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 . This solution neutralizes NaOH in the previous lysis step neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in while precipitating the genomic DNA and SDS in an insoluble white, rubbery precipitate. Spin at an insoluble white, rubbery precipitate. Spin at top speed 1 min. top speed 1 min. n n6. Transfer supernatant to new tube, being 6. Transfer supernatant to new tube, being careful not to pick up any white flakes. careful not to pick up any white flakes. Precipitate the nucleic acids with 0.5mL of Precipitate the nucleic acids with 0.5mL of isopropanol on ice for 10 minutes and centrifuge isopropanol on ice for 10 minutes and centrifuge at top speed for 1 minute.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 (10 mM Tris-Cl, 1 mM EDTA, pH 7.5). Add 10ul of mM Tris-Cl, 1 mM EDTA, pH 7.5). Add 10ul of RNAse A solution (20 mg/ml stock stored at -20 RNAse A solution (20 mg/ml stock stored at -20 C), vortex and incubate at 37 C for 20 to 30 C), vortex and incubate at 37 C for 20 to 30 minutes to digest remaining RNA. minutes to digest remaining RNA. n n8. Extract proteins from the plasmid DNA using 8. Extract proteins from the plasmid DNA using PCIA (phenol/chloroform/isoamyl alcohol) by PCIA (phenol/chloroform/isoamyl alcohol) by adding about 0.3 ml. Vortex vigorously for 30 adding about 0.3 ml. Vortex vigorously for 30 seconds. Centrifuge at full speed for 5 minutes seconds. Centrifuge at full speed for 5 minutes at room temperature. Note organic PCIA layer at room temperature. Note organic PCIA layer will be at the bottom of the tube. will be at the bottom of the tube. n n9. Remove upper aqueous layer containing the 9. Remove upper aqueous layer containing the plasmid DNA carefully avoiding the white plasmid DNA carefully avoiding the white precipitated protein layer above the PCIA layer, precipitated protein layer above the PCIA layer, transferring to a clean 1.5 ml epindorf tube. transferring to a clean 1.5 ml epindorf tube. n n10. Add 100 10. Add 100 m ml of 7.5 M ammonium acetate l of 7.5 M ammonium acetate solution and 1 ml of absolute ethanol to solution and 1 ml of absolute ethanol to precipitate the plasmid DNA on ice for 10 precipitate the plasmid DNA on ice for 10 minutes. Centrifuge at full speed for 5 minutes minutes. Centrifuge at full speed for 5 minutes at room temperature. at room temperature. n n11. Aspirate off ethanol solution and resuspend 11. Aspirate off ethanol solution and resuspend or dissolve DNA pellet in 50ul of DNA. Dissolve or dissolve DNA pellet in 50ul of DNA. Dissolve 5uL in 995ul of water, and spec (blank 5uL in 995ul of water, and spec (blank spectrophotometer to water). The absorbance at spectrophotometer to water). The absorbance at 260 nm multiplied by ten is the concentration of 260 nm multiplied by ten is the concentration of the DNA in units of mg/ml for a 1 cm pathlength the DNA in units of mg/ml for a 1 cm pathlength cuvette (i.e. 50 cuvette (i.e. 50 m mg/ml/OD 260nm). g/ml/OD 260nm). Fig.2. egl4 gene was amplified by RT-PCR图图2. RT-PCR扩增基因扩增基因1.内切葡萄糖苷酶基因内切葡萄糖苷酶基因2.DNA 标准分子量标准分子量