Section G: Gene manipulation

Section G: Gene manipulation
分子生物学研究方法分子生物学研究的内容生物大分子结构和功能：A, B, C, D, E, F 分子生物学研究方法：G, H, I, J 基因表达与调控：L, M, N, O, P, Q 其他(病毒基因组等)：R, S 分子生物学研究方法包括： Section G: Gene Manipulation (基因操作技术) Section H: Cloning Vectors (克隆载体) Section I: Gene Libraries (基因文库) Section J: Analysis of cloned DNA (克隆DNA的分析) Section G: Gene manipulation 华中师范大学生科院杨旭

Section G Gene Manipulation (基因操作技术) G1 DNA cloning: an overview G2 Preparation of plasmid DNA G3 Restriction enzymes and electrophoresis G4-1 Ligation G4-2 Transformation G4-3 Analysis of recombinants Section G: Gene manipulation 华中师范大学生科院杨旭

DNA cloning: 基本步骤以plasmid (vector) 和 E. coli (host) 为例 1. 外源性DNA片段的获取 2. DNA重组: 外源DNA片段与载体分子的体外连接 3. 转化: 将重组DNA导入它们能够复制的宿主细胞 4. 克隆基因的分离与鉴定: Overview G1 外源性DNA G2 & G3 DNA重组 G4-1 转化细胞 G4-2 克隆鉴定 G4-3 Section G: Gene manipulation 华中师范大学生科院杨旭

G1 DNA Cloning: An Overview (DNA 克隆概述)
DNA cloning (DNA克隆) Sub-cloning (亚克隆) DNA libraries (DNA文库) Screening libraries (筛选文库) Vectors (载体) Plasmids (质粒) Hosts (宿主) Analysis of clone (克隆分析) Section G: Gene manipulation 华中师范大学生科院杨旭

DNA cloning: （DNA克隆的定义）
Definition: It is the making process to place the target (目的) gene (the gene of interest) in a vector (an autonomously (自主) replicating piece of DNA), forming recombinant DNA, which then is placed into another host species. DNA克隆：在分子生物学中，人们把外源性DNA插入具有复制能力的载体DNA中，形成一种重组DNA，再将这种重组DNA转化到宿主细胞中，使之得以永久保存和复制的过程称为“DNA克隆”、“基因克隆”或简称“克隆”。 E S target gene Host transformant 抗生素抗性基因 vector Ori 克隆：原意为“无性增殖” Section G: Gene manipulation 华中师范大学生科院杨旭

DNA cloning: 基本步骤以plasmid (vector) 和 E. coli (host) 为例 1. 外源性DNA片段的获取 (1) Preparation of plasmid DNA containing the cloned target gene. (2) Digestion of the plasmid with restriction endonucleases. (3) Separation of the fragments by agarose gel electrophoresis. (4) Purification of the desired target fragment. 2. DNA重组: 外源DNA片段与载体分子的体外连接 (5) Ligation of the fragments, to form a new recombinant molecule. 3. 转化: 将重组DNA导入它们能够复制的宿主细胞 (6) Transformation of the ligated plasmid into an E. coli strain (株). 4. 克隆基因的分离与鉴定: (7) Selection of transformed bacteria (see Topic G4). (8) Analysis of recombinant plasmids (see Topic G4). Section G: Gene manipulation 华中师范大学生科院杨旭

Subcloning (亚克隆：从载体到载体)
Definition: It is the process to transfer of a fragment of cloned DNA from one vector to another. Experimental steps: 1. Preparation: of the plasmid 2. Digestion of the plasmid 3. Separation of the fragments 4. Purification of target fragment 5. Ligation of the fragments 6. Transformation 7. Selection of transformed bacteria. 8. Analysis of plasmids. + - 质粒分析 Section G: Gene manipulation 华中师范大学生科院杨旭

DNA libraries (DNA文库: 外源DNA的来源)
Definition: DNA libraries are sets of DNA clones (vectors/hosts), each of which has been derived from the insert of a different fragment into a vector followed by propagation (增殖) in the host. Classification and features: 1. Genomic libraries: They are derived from random fragments of DNA from the genomes of species by shotgun approach ( 鸟枪法); The approach may be an inefficient of finding a gene, especially in eukaryotic genomes, where much of the DNA is noncoding. Section G: Gene manipulation 华中师范大学生科院杨旭

DNA libraries (DNA文库: 外源DNA的来源)
2. cDNA libraries: (ProteinCellmRNAcDNAVectorHost) They are derived from the mRNA by reverse transcription and are then inserted into a vector; cDNA libraries are efficient for finding and cloning a gene, but only the coding region, not the surrounding genomic sequences. Section G: Gene manipulation 华中师范大学生科院杨旭

Screening libraries (筛选文库: 寻找目的基因)
Definition: It is the process to use a DNA probe to find the clone that contains the gene of interest. DNA probe: It is a radioactively labeled short DNA sequence which is partially complementary to a region of the target gene sequence, therefor, the target gene or clone can be detected by its hybridization. Making of the probes: The probe sequence might be an oligo-nucleotide (寡核苷酸) derived from the sequence of the protein product of the gene; From a related gene from another species. An increasingly important method for the generation (生产) of probes is PCR Section G: Gene manipulation 华中师范大学生科院杨旭

Vectors-I (载体的定义和特征) 定义: 具有自主复制能力的DNA分子就是所谓的分子克隆的载体，质粒、噬菌体、病毒和人工染色体等小分子量的复制子都可以作为导入基因的载体。 Section H 将专门讲解用于基因克隆的载体 The features of vectors: Vectors must normally be capable of being replicated and isolated independently of the host's genome; Vectors also have a selectable marker, a gene which allows host cells conferring resistance to a toxin. There are some vectors, for example phage  (see Topic H2), which can incorporate DNA into the host genome for longer term expression of cloned genes. Section G: Gene manipulation 华中师范大学生科院杨旭

Vectors-II (载体的种类) The common vectors: 1. Plasmids: Circular plasmid of E. coli: used in E. coli (host); Yeast episomal (游离型) plasmids: used in yeast; Agrobacterium tumefaciens (根瘤农杆菌) Ti plasmid: in plant. 2. Bacteriophages (viruses infecting bacteria; see Topic R2): Phage : also been used in E. coli, for cloning larger fragments Phage M13: used to clone ssDNA used in E. coli. 3. Cosmids (粘粒plasmid-bacteriophage hybrids): (see Topic H3). 4. Artificial chromosomes: for cloning huge fragments from humans. BAC: Bacterial artificial chromosomes (in E. coli); YAC: Yeast artificial chromosomes (in Yeast). 5. Virus: for other eukaryotic cells in culture (培养状态) SV40: Simian virus 40 (猴空泡病毒40); Retroviruses. (see Topic H4). Section G: Gene manipulation 华中师范大学生科院杨旭

Plasmids (最常见的载体--质粒)
The first cloning vectors to be used, in the mid 1970s, were natural plasmids originally from E. coli. Structure and features: Plasmids are  small in size, from 2 to around 200 kb  extrachromosomal circular molecules  which exist in multiple copies (up to a few hundreds)  within the host E. coli cells.  They contain an origin of replication (ori), which enables them to be replicated independently. Section G: Gene manipulation 华中师范大学生科院杨旭

Plasmids (最常见的载体--质粒)
Resistance gene: They usually carry a few genes, one of which may confer (赋予) resistance to antibacterial substances: 1. The most widely known resistance gene is ampr gene, which encodes the enzyme -lactamase (-内酰胺酶), that degrades penicillin antibiotics such as ampicillin (氨苄青霉素). 2. Another is the tetA gene, which encodes a transmembrane pump able to remove the antibiotic tetracycline (四环素) from the cell. Section G: Gene manipulation 华中师范大学生科院杨旭

Hosts (宿主细胞的定义和种类) Host 的定义：基因克隆的载体或者重组DNA分子，得以复制扩增和保存的细胞，包括原核细胞和真核细胞。单克隆的定义：带有某一种重组DNA的宿主细胞，增殖构成的一群具有遗传一致性的细胞。 The common hosts: E. coli (大肠杆菌): The initial isolation and analysis of DNA fragments is almost always carried out using the E. coli as the host (for circular plasmid, phage , phage M13, cosmid, BAC); Yeast (酵母菌): It is being used to manipulate (制备) very large fragments of the human genome (for episomal plasmid, YAC). 其他细胞，例如: 根瘤农杆菌 (for Ti plamid)、昆虫培养细胞 (for baculovirus 杆状病毒)、哺乳动物培养细胞 (for SV40, retroviruses, shuttle vectors)、枯草杆菌、动物细胞等。 Section G: Gene manipulation 华中师范大学生科院杨旭

Analysis of clone (克隆分析)
Once a clone containing a target gene is identified, the structure of the cloned fragment may be investigated: 1. further using restriction mapping (限制性酶切图谱), the analysis of the fragmentation of the DNA with restriction enzymes (see Topic J1) and by agarose gel electrophoresis using marker of known sizes (see Topic G3), 2. or ultimately (最终) by the sequencing (测序) of the entire fragment (see Topic J2). 3. The sequence can then be analyzed by comparison with other known sequences from data bases, and the complete sequence of the protein product determined (see Topic J2). Section G: Gene manipulation 华中师范大学生科院杨旭

一、外源性DNA片段的获取 G2 Preparation of Plasmid DNA (质粒的制备) Plasmid minipreparation (质粒的小量制备) G3 Restriction Enzymes and Electrophoresis (Digestion, separation and purification) Restriction endonucleases (限制性内切核酸酶) Restriction sequences (限制性识别序列) Cohesive ends (粘末端) Restriction digests (限制性酶解) Agarose gel electrophoresis (琼脂糖凝胶电泳) Section G: Gene manipulation 华中师范大学生科院杨旭

Plasmid mini-preparation (质粒的小量制备)
Bacterial culture (细菌培养) Alkaline lyses (碱裂解) Phenol extraction (酚抽提) Ethanol precipition (乙醇沉淀) Cesium chloride gradient (氯化铯梯度) Section G: Gene manipulation 华中师范大学生科院杨旭

质粒的小量制备流程离心集菌细菌振动培养 1. 悬浮细胞沉淀; 2. 加入溶菌酶； 3. 加去污剂SDS; 4.NaOH (碱裂解) 质粒 RNA 蛋白质蛋白质、染色体DNA、膜酚-氯仿抽提 CsCl梯度离心质粒 RNA 水溶液蛋白质质粒染色体DNA 变性蛋白酚--氯仿 RNA 1. 取水溶液 2. 乙醇沉淀 3. 用RNA酶 1. 取质粒带 2. 乙醇沉淀纯化质粒 Section G: Gene manipulation 华中师范大学生科院杨旭

Digestion: Restriction endonucleases (限制性内切核酸酶)
Function: Restriction-modification systems occur in many bacterial species, and constitute (构成) a defense mechanism against the foreign DNA into the cell. Structure: Restriction-modification system consist of two parts： 1. The first part is a restriction endonuclease, which recognizes a short, symmetrical DNA sequence (Fig. 1), and hydrolyzes the DNA backbone in each strand at a specific site. 2. The second part is a methylase, which adds a methyl group to a C or A base within the same recognition sequences. This modification protects the host DNA against the endonuclease. Section G: Gene manipulation 华中师范大学生科院杨旭

Digestion: Restriction sequences (识别序列)
Definition: It is a short palindromic (回文) sequences, at which restriction enzymes cleave DNA symmetrically (对称地) in both strands. Acting Steps: EcoRI as an example. Recognizing: The restriction endonucleases EcoR I acts as a dimer, will only recognize a 6 bp palindromic (回文) sequence. Cutting: The product of the cutting reaction is two restriction fragments, each with a 5'-end with a phosphate group and a 3'-end with a free hydroxyl group. EcoR I Recognizing 5’-GAATTC-3’ 3’-CTTAAG-5’ Cutting 5’-G-OH P-AATTC-3’ 3’-CTTAA-P HO-G-5’ annealing 5’-GAATTC-3’ 3’-CTTAAG-5’ Section G: Gene manipulation 华中师范大学生科院杨旭

Digestion: Cohesive ends (粘末端)
Definition: Some of the products of restriction enzyme digestion have protruding (突出的) ends, and these ends are known as cohesive, or 'sticky' ends. Features: Those products of restriction enzyme digestion with protruding ends have a further property:   They can bind to any other end with the same overhanging (突出的) sequence, by base pairing (annealing 退火配对) of the single-stranded tails.  For example, any fragment formed by an EcoR I cut can anneal to any other fragment formed in the same way, and may subsequently be joined covalently by ligation.  In fact, in some cases, DNA ends formed by enzymes with different recognition sequences may be compatible (兼容) Section G: Gene manipulation 华中师范大学生科院杨旭

Digestion: Restriction digests-I (限制性酶解)
Application: Digestion of plasmid or genomic DNA is carried out with restriction enzymes for analytical or cloning preparation purposes. Examples: The digestion of a sample plasmid with two different restriction enzymes, Bam HI and EcoR I. E B Plasmid with gene X E B EcoRI BamHI B Section G: Gene manipulation 华中师范大学生科院杨旭

Digestion: Restriction digests-II (限制性酶解)
Reaction system: All restriction enzymes require Mg2+ (magnesium) usually at a concentration of up to 10 mM; but different enzymes require different :  pHs,  NaCl concentrations or  other solution constituents (成份). Reaction process: The DNA is  incubated at the  optimum temperature (37C) with  the enzyme and  the appropriate buffer, in a volume of perhaps 20 l.  A dye mixture is then added to solution, and  the sample is loaded (上样) on to an agarose gel. Section G: Gene manipulation 华中师范大学生科院杨旭

Separation: Agarose gel electrophoresis-I (琼脂糖凝胶电泳)
Definition: Agarose is a polysaccharide derived from seaweed, which forms a solid gel (固体凝胶) when dissolved in aqueous solution at concentrations between 0.5 and 2% (w/v). Principle (原理): Movement: When an electric field is applied to an agarose gel in the presence of a buffer solution, DNA fragments move through the gel towards the + electrode, because DNA negatively charged. Movement rate: The movement rate is dependent on fragment sizes and shapes of the DNA and the electrophoresis may be used to separate mixtures of DNA fragments by the rate. + - DNA sample 电导缓冲液 Migration of DNA Agarose gel Section G: Gene manipulation 华中师范大学生科院杨旭

Separation: Agarose gel electrophoresis-II (琼脂糖凝胶电泳) General procedures: The DNA samples are placed in wells (上样孔) in the gel surface. the power supply is switched on and the DNA is allowed to migrate (迁移) through the gel in separate lanes or tracks (泳道). The added dye (EB) also migrates, and is used to find the progress. The DNA is stained by the EB in the gel, the DNA shows up as an orange band on illumination (光照) by UV light. Section G: Gene manipulation 华中师范大学生科院杨旭

Separation: Agarose gel electrophoresis-III (琼脂糖凝胶电泳) Plasmid with gene X E B Result reading: see p103 Fig 4 Tracks M: A set of linear marker DNA fragments of known sizes; Track U: Undigested plasmid DNA consists of two bands. The lower band consists of negatively supercoiled plasmid DNA. The upper band is the nicked (缺口) DNA, which has lower mobility; Track B: digestion with BamHI, containing a single fragment; Track E: digestion with EcoRI, containing five fragments, the third largest fragment contains the gene X, which could be purified from the gel and ready for ligation into a new vector. Section G: Gene manipulation 华中师范大学生科院杨旭

琼脂糖及聚丙烯酰胺分辩DNA片段能力 Section G: Gene manipulation 华中师范大学生科院杨旭

Purification of fragments (片段的纯化)
Purposes: Agarose gels may also be used preoperatively to isolate specific fragments for use in subsequent ligation and other cloning experiments. Purification method: Fragments are cut from the gel, and treated by one of a number of procedures to purify the DNA away from the contaminating agarose. Example: If we assume that the EcoRI fragment, which contains the gene X, is the target DNA for a subcloning experiment, then the third largest fragment (in track E of Fig. 4a) could be purified from the gel, ready for ligation into a new vector (see G4). Section G: Gene manipulation 华中师范大学生科院杨旭

+ - 质粒分析 Section G: Gene manipulation 华中师范大学生科院杨旭

二、DNA 重组外源DNA片段与载体分子的体外连接 G4-1 Ligation (DNA Recombination)
DNA ligation (DNA连接) Recombinant DNA molecules Alkaline phosphatase (碱性磷酸酶) Section G: Gene manipulation 华中师范大学生科院杨旭

DNA ligation (DNA连接) Purpose: To insert a target DNA fragment into a vector. Function of DNA ligases: they will repair (ligate) a break in one strand of a dsDNA molecule. Energy sources: DNA ligase from E. coli uses NAD+ as energy source; T4 DNA ligase uses ATP (more commonly used). Different ends: Cohesive ends: Ligases are efficient at sealing the broken phosphodiester bonds for cohesive ends, Blunt ends: just T4 ligase can even ligate one blunt end to another, but with rather lower efficiency. Section G: Gene manipulation 华中师范大学生科院杨旭

Recombinant DNA molecules-I (重组DNA分子)
Making of the recombinant DNA: Target DNA:  may be a single fragment isolated from agarose gel, or  a mixture of many fragments from genomic DNA. Vector DNA: the new vector DNA can be cut with the same enzyme. If the target DNA has been prepared by digestion with same enzyme (EcoRI). Recombinant molecules: The products are circular molecules with the target fragment inserted at the EcoRI site of the vector molecule with either orientation (方向). E S E S 抗生素抗性基因 Ori E E EcoRI A E B Section G: Gene manipulation 华中师范大学生科院杨旭

Recombinant DNA molecules-II (重组DNA分子)
A problem: The recreation of the original vector plasmid, by circularization of the linear vector alone, is a competing side reaction which can make problems on the recombinant identification. Solution one--Paired distinct restriction: One solution is to prepare both the target DNA and the vector DNA using a pair of distinct restriction enzymes, such that they have non-compatible cohesive ends at either end. The likelihood (可能性) of ligating the vector into a circle is then much reduced. Section G: Gene manipulation 华中师范大学生科院杨旭

Alkaline phosphatase (碱性磷酸酶)
Solution two: Linear vector: Alkaline phosph-atase removes phosphate groups from the 5'-ends of DNA molecules. The linear vector will hence be unable to ligate into a circle, since no phosphates are available. Target DNA: A ligation with a target DNA insert can still proceed, since one phosphate is present to ligate one strand at each cut site. The nicks in the other strands will be repaired when transformated in E.coli. E 碱性磷酸酶 E S G -OH CTTAA -OH HO -AATTC HO -G HO -AATTC HO -G G -OH CTTAA -OH P Section G: Gene manipulation 华中师范大学生科院杨旭

三、转化: 将重组DNA导入它们能够复制的宿主细胞 G4 -2 Transformation (转化)
Selection of plates (平板选择培养法) Transformation efficiency (转化率) Growth and storage of transformants (转化子增殖和保存) Section G: Gene manipulation 华中师范大学生科院杨旭

Transformation-I (转化)
Definition: It is the process of take-up (吸收) of foreign DNA (normally plasmids) by bacteria. Plasmids are cloned (无性繁殖) by transferring into E. coli with defined genetic properties. Purpose: The recombinant and other plasmid formed by ligation must be  isolated from one another and  replicated by a host. Hosts: The most common hosts are strains of special E. coli, which must not express a restriction-modification system. The host cell can accept only one kind of foreign plasmid. Competent cells (感受态细胞): It was discovered that E. coli cells treated with solutions containing Ca2+ ions were rendered (表达出) susceptible (易感性) to take up (吸收) exogenous DNA. Cells pretreated with Ca2+, in order to render them able to take up DNA, are known as competent cells (商品化的更好用). Section G: Gene manipulation 华中师范大学生科院杨旭

Transformation-II (转化)
Transformation process: Taking up DNA: A solution of a plasmid molecule is combined with a suspension (悬浮液) of competent cells for a period (30-40min), to allow the DNA to be taken up. Recovering: The mixture is then heat-shocked (热激) at 42 ºC for 1-2 min. This step induces enzymes involved in the repair of DNA, which allow the cells to recover from unusual conditions of the transformation process, and increases the efficiency. Transformation: The cells are then incubated in a growth medium and finally spread on an agar plate and incubated until single colonies of bacteria grow. plasmid 感受态细胞 Taking up DNA Recovering Transformation Section G: Gene manipulation 华中师范大学生科院杨旭

Selection of plates (平板选择培养法)
A problem: If all the competent cells present in a transformation reaction were allowed to grow on an agar plate, then many thousands or millions of colonies would result. Hence, a method for the selection of clones containing a plasmid is required. Principle: This is almost always provided by the presence of an antibiotic resistance gene on the plasmid vector, for example the -lactamase gene (ampr) conferring resistance to ampicillin. If the transformed cells are grown on plates containing ampidllin, only those cells which are expressing -lactamase (due to the presence of a transformed plasmid) will survive and grow. Notice: If a ligation mixture (but not a single plasmid molecules) had been used for the transformation, we would not know at this stage which clones contain recombinant plasmids with a target fragment incorporated. Section G: Gene manipulation 华中师范大学生科院杨旭

Transformation efficiency (转化率)
Definition: The quality of competent cells may be measured by determining the transformation efficiency, defined as the number of colonies (菌落数) formed per g pure plasmids. Range and usage: Transformation efficiencies can range: 103 colonies/g plasmids: can be used for crude transformation protocols, which would only be appropriate for transferring an intact (完整) plasmid to a new host strain (subcloning); 105 colonies/g plasmids: would be enough for a simple cloning experiment of the kind outlined in this book. >108 colonies/g plasmids: can be used for generation of libraries; Section G: Gene manipulation 华中师范大学生科院杨旭

Growth and storage of transformants
Method: Single colonies from a transformation plate are trans-ferred to broth and grown overnight until the stationary phase. Notice: The broth (培养液) must be included with the antibiotic, and used to select the transformants on the original plate, to maintain the selection for the presence of the plasmid. Storage: Method: It is normal practice to prepare a stock of each culture at this stage, by freezing a portion (部分) of the culture in the presence of glycerol (甘油，或者二甲亚砜). Reason: The effect of glycerol stock (贮存液) is to protect the cells from ice crystal formation. The stock will enable the same strain/plasmid to be grown and prepared again if and when it is required. Section G: Gene manipulation 华中师范大学生科院杨旭

四、克隆基因的分离与鉴定: G4 -3 Analysis of Recombination
Screening transformants (筛选转化子) Gel analysis (凝胶分析) Fragment orientation (插入片段定向) Section G: Gene manipulation 华中师范大学生科院杨旭

Screening transformants (筛选转化子)
Transformant definition: It is the cloned bacteria which contain a recombinant plasmid, with inserted target fragment. Purpose: Once a set of transformant clones has been produced in the experiment, the first requirement is to know which clones contain a recombinant plasmid, with inserted target fragment. Methods: 1. In the case of a simple subcloning experiment, the normal method of screening is to prepare (提取) the plasmid DNA from a number of clones and analyze it by agarose gel electrophoresis. 2. Commercial plasmids have been designed to facilitate this process, and are described in Topic H1. 3. In many cases, such as the screening of a DNA library, it will then be necessary to identify the interest clone among thousands or even hundreds of thousands of others (Topic I3). Section G: Gene manipulation 华中师范大学生科院杨旭

+ - Gel analysis (凝胶分析) Application: Simple gel analysis: Recombinant plasmids can usually be simply distinguished from recreated (重组) vectors by the relative sizes of the plasmids, and Gel analysis of restriction digests: Tracks corresponding to the vector plasmid and to recombinants are indicated. Tracks U: The larger size of the recombinant plasmid is seen by comparing the undigested plasmid samples, containing super-coiled and nicked bands (see Topic G3); Track E: The excision (切割产物) from the recombinant is seen in the EcoRI digest. Section G: Gene manipulation 华中师范大学生科院杨旭

Fragment orientation (插入片段的定向) 插入方向正确有意链模板链启动子基因插入方向错误有意链模板链启动子 Section G: Gene manipulation 华中师范大学生科院杨旭

Fragment orientation (插入片段的定向)
Orientation: The orientation of the fragment can be determined using a restriction digest with: An enzyme (SalI) which is known to cut asymmetrically (不对称地) within the insert sequence, together with Another enzyme (HindIII) which cuts at some specified site in the vector. Illustration: This is illustrated using a double digest with: The patterns expected from two orientations, A and B, of inserted fragment are illustrated in Fig. 4 (tracks H/S). 小大 A H S B H S Section G: Gene manipulation 华中师范大学生科院杨旭

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Section G: Gene manipulation 华中师范大学生科院杨旭

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