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1、基因工程的操作流程,基因工程的操作包含以下步骤：获得目的基因 构造重组 DNA 分子 转化或转染 表达 蛋白质产物的分离纯化,第二讲目的基因的制备,需要克隆的DNA片段,编码某种蛋白质,研究某基因结构和功能的关系,研究某基因与疾病的关系,一、从基因文库中获得目的基因,二、用PCR方法获得目的基因,三、基因的化学合成,一、从基因文库中获得目的基因,A gene library,also called genomic library,is a population of host bacteria,each of which carries a DNA molecule that was inse
2、rted into a cloning vector,such that the collection of cloned DNA molecules represents the entire genome of the source organism.A gene library is also called gene bank.,1、基因文库（Gene library）,基因文库，又叫基因组文库，系指在一种载体分子中随机地克隆着某种生物、组织、器官或细胞类型的所有的DNA片段而构成的克隆集合体。在理想的情况下，它应包含有该生物种全部的遗传信息。,DNA文库(DNA library)，基因
3、文库是在基因工程诞生的早期就已被使用，已经习惯且通俗易懂。但近年来一般称基因文库为DNA文库，这是一种更加符合实际更加科学的名称。因为基因文库从本质上讲是由来自染色体基因组的全部DNA片段组成的。基因组文库(Genomic library)，系指生物体基因组DNA经过核酸限制内切酶作部分切割之后，克隆在适当的载体分子上，然后将这重组的混合物转化给宿主菌株(例如大肠杆菌)，便构成了包含该生物体整个基因组全部遗传信息的基因组文库。,The DNA molecules of an organism in interest are isolated.The DNA molecules are then
4、 partially digested by an endonuclease restriction enzyme.Sometimes,the DNA molecules are digested to different lengths of time in order to ensure that all the genes have been digested to manageable sizes.The digested DNA molecules are run on agarose electrophoresis for which a suitable range of len
5、gths of DNA pieces are isolated and ligated to vector plasmids.Statistically,99%of the genes will be incorporated into the plasmids.The plasmids then can be taken up by suitable hosts.,基因文库的构建（Creating a library）,The hosts are kept in liquid media and can be frozen at-80C for a long period of time.U
6、sually the hosts are bacteria that do not contain any plasmids so as to be sensitive to antibiotics.The process of subdividing genomic DNA into clonable element and inserting them into host is called creating a library,a clone bank or a gene bank.A complete library of host cell will contain all of t
7、he genomic DNA of the source organism.,制备全部基因组的DNA片断机械断裂法用超声波或高速搅拌DNA溶液，断裂片段两端没有与克隆载体匹配的粘末端,因此插入载体之前还需要进行修饰加工，少用限制性内切酶降解（鸟枪法）过去用EcoR,现在用Sau3A，断裂片段两端有与克隆载体匹配的粘末端，可以直接与处理过的载体连接。,构建基因组文库全过程,分离纯化基因组DNA 提取哺乳动物细胞染色体DNA（酚抽提法）,DNA片段与载体的连接包装常用载体：粘性质粒，噬菌体，酵母人工染色体基因组DNA+粘性质粒重组DNA转化细菌基因组DNA+噬菌体 重组DNA包装成噬菌体感染细菌
8、1个克隆含有基因组的某个片段，整个克隆群体就包含基因组的全部基因片段总和。,实际克隆数 1975年L.Clarke等提出了一种计算完整的基因组文库最低重组体所需实际克隆数（N）的公式：N=ln(1-P)/ln(1-f)N：文库必需的克隆数 P：文库中目的DNA片段的出现概率 f：插入片段大小/全基因组大小的比值,基因组文库的大小,理论克隆数文库克隆数=基因组DNA总长/DNA插入片段平均长,从人基因组DNA（总长度为3109bp）的文库中筛选到长约1.5kb目的基因的可能性为99，那么这个基因组文库要多大？理论克隆数：3109/1.5 1032106In（10.99）实际克隆数：N=9.210
9、6 In（11.51033109）,用质粒（pBR322）克隆目的基因（1.5kb）需要有9106克隆才能汇集人基因组全部DNA序列;若 用噬菌体载体可构建含15kb目的基因的人基因组DNA文库，按上式计算所需要的重组体克隆数可以减少到9105,而要用柯斯质粒将40kb目的基因片断所需要重组体克隆数可降到3.5105左右,均可满足建库要求。,三种常用基因组文库克隆载体的比较,对细菌、酵母和真菌来讲，一个完整的基因组文库所需的克隆数还是在可操作的范围内的。但植物和动物的一个完整的基因组文库包含了太多的不同克隆，如果从中鉴定出目的克隆，工作量是相当大的。这时可以构建针对某一种细胞被表达的基因的基因
10、文库(cDNA文库),cDNA(Complementary DNA),In genetics,complementary DNA(cDNA)is DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase.cDNA is often used to clone eukaryotic genes in prokaryotes.,The central dogma of molecular biology outlines that in synthe
11、sizing proteins,DNA is transcribed into mRNA,which is translated into protein.One difference between eukaryotic and prokaryotic genes is that eukaryotic genes can contain introns(intervening sequences),which are not coding sequences,and must be spliced out of the RNA primary transcript before it bec
12、omes mRNA and can be translated into protein.Prokaryotic genes have no introns,so their RNA is not subject to splicing.,Often it is desirable to express eukaryotic genes in prokaryotic cells.A simplified method of doing so would include the addition of eukaryotic DNA to a prokaryotic host,which woul
13、d transcribe the DNA to mRNA and then translate it to protein.However,as eukaryotic DNA has introns,and since prokaryotes lack the machinery to splice them,the splicing of eukaryotic DNA must be done prior to adding the eukaryotic DNA into the host.This DNA which was made as a complementary to the R
14、NA is called complementary DNA(cDNA).To obtain expression of the protein encoded by the eukaryotic cDNA,prokaryotic regulatory sequences would also be required(e.g.a promoter).,cDNA无内含子,长度较基因组基因小,使操作更为方便。mRNA比基因组中基因少,因此筛选某一特定功能基因工作量较小。mRNA中拷贝数大于基因组中的拷贝数,利于获得较多的模板。可在原核细胞中表达有生物活性蛋白 不同种类不同状态的细胞的cDNA文库不
15、同不能研究基因组的结构功能,cDNA文库特点,基因组文库是含有某种生物体全部基因的随机片段的重组DNA克隆群体；cDNA文库是指含有所有重组cDNA的克隆群体。基因组文库：来源于基因组DNA，反映基因组的全部信息，用于基因组物理图谱的构建，基因组序列分析，基因在染色体上的定位，基因组中基因的结构和组织形式等。cDNA文库：来源于细胞表达出的RNA，反映基因组表达的基因序列信息，用于研究特定细胞中基因的表达状态和表达基因的功能等。,比较,cDNA文库(cDNA library)，将纯化的某种生物的特定发育阶段或组织的mRNA，经反转录酶作用合成双链的DNA群体，同适当的载体分子重组之后转化给寄主
16、菌株，如此便构成了特定的cDNA文库。这种文库与基因组文库不同，它只包括特定的发育阶段特定组织或器官中表达的基因，而不是全部的基因。,cDNA文库(cDNA library)的构建,生物体是由很多不同类型的细胞组成的，如神经细胞、血细胞、肝细胞等。每种细胞都含有相同数目基因，但不同类型的细胞所表达的基因是有差别的。因为在任何一种细胞中都只有部分基因得到表达，我们可以利用这一事实来制备cDNA文库：制作文库的起始材料不再是DNA而是信使RNA（mRNA）。由于只有那些被表达的基因会被转录成mRNA，因此以mRNA为材料所得到的克隆将只包含细胞所有基因的一部分。,当目的基因在某一细胞类型中高水平表
17、达时，建立该细胞的cDNA文库就特别有利于目的基因的分离。麦醇溶蛋白是小麦中的一种重要的营养蛋白，在发育的小麦种子中，编码醇溶蛋白的基因高水平表达。这些细胞总mRNA的30%是麦醇溶蛋白的mRNA。显然，如果我们从小麦种子中克隆mRNA，将会得到大量特异表达的麦醇溶蛋白基因的克隆。,cDNA文库的构建方法,cDNA克隆的基本过程是在一系列酶的作用下，使总poly(A)RNA制剂转变成双链cDNA群体，并插入到适当的载体中，然后再转化大肠杆菌细胞。如此便构建了包含所有编码序列的cDNA文库。此种技术，已成为当今研究分子生物学的基本手段。,在真核细胞中，大部分mRNA的3末端都具有poly(A)尾
18、。,oligo(dT)can be bound to the poly(A)tail and used to recover the mRNA.,(AAAAAAAAAA)n,5 cap,原理,（1）分离细胞总RNA，然后从中纯化出poly(A)RNA,通过寡聚（dT）纤维素柱 将结合着（dT）的磁珠加入到总RNA中，再用磁铁收集磁珠，最后洗出mRNA。,（2）合成第一链cDNA,oligo(dT)与mRNA的poly(A)结合可以作为合成第一链cDNA的引物.在逆转录酶的作用下，以mRNA为模板，以oligo(dT)为引物模板合成cDNA，形成RNADNA杂交分子。这种合成第一链cDNA的方法
19、又称为利用oligo(dT)引导的cDNA合成法。,置换合成法 该方法利用第一链在反转录酶作用下产生的cDNA:mRNA杂交链不用碱变性,而是在dNTP存在下,利用RNA酶H在杂交链的mRNA链上造成切口和缺口。从而产生一系列RNA引物,使之成为合成第二链的引物,在大肠杆菌DNA聚合酶的作用下合成第二链。,（3）合成双链cDNA分子,该反应有3个主要优点:(1)非常有效;(2)直接利用第一链反应产物,无须进一步处理和纯化;(3)不必使用S1核酸酶来切割双链cDNA中的单链发夹环。目前合成cDNA常采用该方法。,自身引导法 合成的单链cDNA 3端能够形成一短的发夹结构,这就为第二链的合成提供了
20、现成的引物,当第一链合成反应产物的DNA:RNA杂交链变性后利用大肠杆菌DNA聚合酶 Klenow片段cDNA第二链,最后用对单链特异性的S1核酸酶消化该环,即可进一步克隆。,但自身引导合成法较难控制反应，而且用S1核酸酶切割发夹结构时无一例外地将导致对应于mRNA 5端序列出现缺失和重排,因而该方法目前很少使用。,聚合酶,（4）将合成的双链cDNA分子重组到质粒载体或噬菌体载体上，导入到大肠杆菌细胞增殖。,如何从文库中获得基因？,Nucleic acid hybridization is the process,discovered by Alexander Rich,of combinin
21、g complementary single-stranded nucleic acids into a single molecule.Nucleotides will bind to their complement under normal conditions,so two perfectly complementary strands will bind to each other readily.This is called annealing.Annealing may be reversed by heating the double stranded molecule of
22、DNA(or RNA or DNA:RNA)to break the hydrogen bonds between bases and separate the two strands.This is called melting or denaturation.,核酸杂交技术,核酸杂交（nucleic acid hybridization），它是指两个互补或部分互补的核酸分子形成稳定的碱基配对杂交体的过程。,核酸分子单链之间有互补的碱基顺序，通过碱基对之间非共价键（主要是氢键）的形成即出现稳定的双链区，这是核酸分子杂交的基础。杂交分子的形成并不要求两条单链的碱基顺序完全互补，所以不同来源的核
23、酸单链只要彼此之间有一定程度的互补顺序（即某种程度的同源性）就可以形成杂交双链。分子杂交可在DNA与DNA、RNA与RNA或RNA与DNA的二条单链之间进行。,如果用与目的基因互补的DNA或RNA作探针，就可以利用核酸杂交技术(nucleic acid hybridization)鉴定出特定的重组体。,由于DNA一般都以双链形式存在，因此在进行分子杂交时，应先将双链DNA分子解聚成为单链，这一过程称为变性，一般通过加热或提高pH值来实现。使单链聚合双链的过程称为退火或复性。用分子杂交进行定性或定量分析的最有效方法是将一种核酸单链用同位素或非同位素标记成为探针，再与另一种核酸单链进行分子杂交。,
24、There exist two kinds of nucleic acid hybridization.Both are methods in which radioactively labeled single DNA strands of known base sequences are used as a probe to detect the nucleotide sequence of another single stranded DNA or RNA molecule.The first one is widely used for detection of specific g
25、enes in cellular DNA.It was developed by E.M.Southern and is called Southern Blotting.,In this procedure,the target DNA is digested with one or more restriction endonucleases,size-fractionated by agarose gel electrophoresis,denatured and transferred to a nitrocellulose or nylon membrane for hybridiz
26、ation.During the electrophoresis,DNA fragments,which are negatively charged because of the phosphate groups,are repelled from the negative electrode towards the positive electrode,and sieved through the porous gel.,Smaller DNA fragments move faster.For fragments between 0.1 and 20 kb long,the migrat
27、ion speed depends on fragment length,but scarcely at all on the base composition.Thus,fragments in this size range are fractionated by size in a conventional agarose gel electrophoresis system.To achieve efficient size-fractionation of large fragments(40 kb to several megabases),a more specialized s
28、ystem is required,such as a pulsed-field gel electrophoresis apparatus.,Following electrophoresis,the test DNA fragments are denatured in strong alkali.As agarose gels are fragile,and the DNA in them can diffuse within the gel,it is usual to transfer the denatured DNA fragments by blotting on to a d
29、urable nitrocellulose or nylon membrane,to which single-stranded DNA binds readily.The individual DNA fragments become immobilized on the membrane at positions which are a faithful record of the size separation achieved by agarose gel electrophoresis.,Subsequently,the immobilized single-stranded tar
30、get DNA sequences are allowed to associate with labeled single-stranded probe DNA.The probe will bind only to related DNA sequences in the target DNA,and their position on the membrane can be related back to the original gel in order to estimate their size.,印迹法（blot）的主要步骤：（1）DNA 用限制性内切酶处理。（2）DNA 片段混
31、合物通过电泳分离。（3）电泳后，通过印迹技术转到酯酰 纤维薄膜上，以便操作。（4）用已知小片段DNA 作为探针，互补 结合需要找的基因片段。（5）探针DNA 片段已用放射性元素标记，使胶片感光后可看出。,The second method is called northern blotting,it is used to identify the base pair sequence of RNA.RNA is extracted and fractionated according to size by electrophoresis.Then the procedure is the sa
32、me as in Southern Blotting.Northern blotting is frequently used in studies of gene expression(e.g.whether specific mRNA is present in different types of cells),(AE)Procedure for Northern blotting.(A)RNA is isolated from various tissues and is separated by size using gel electrophoresis.(B)The gel is
33、 then placed on a paper wick,which absorbs an ionic solution from a trough.(C)A filter that traps RNA is placed above the gel,and blotting paper is placed above the filter.Capillary action draws the solution through the gel,trapping the RNA on the filter.(D)The filter is incubated with radioactive s
34、ingle-stranded DNA complementary to the mRNA of interest.(E)After any unbound DNA is washed off,autoradiography localizes the mRNA in the samples that contain it.(F)Drawing of a developmental Northern blot showing the presence of Pax6 mRNA in the eye,brain,and pancreas of a mammalian embryo.,用于菌落和噬菌
35、斑的杂交技术,重组DNA分子无论是存在于菌落中还是存在于噬菌斑中，都可以用探针杂交技术鉴定出来。首先把菌落或噬菌斑转移到硝酸纤维膜或是尼龙膜上，裂解，使DNA分子变性，80加热，使单链DNA分子牢牢地结合到硝酸纤维膜或是尼龙膜上。DNA是通过它们的磷酸核糖骨架与膜结合，它们的碱基是自由的，可以和互补的核酸分子配对。,用于菌落和噬菌斑的杂交技术,重组DNA分子无论是存在于菌落中还是存在于噬菌斑中，都可以用探针杂交技术鉴定出来。首先把菌落或噬菌斑转移到硝酸纤维膜或是尼龙膜上，裂解，使DNA分子变性，80加热，使单链DNA分子牢牢地结合到硝酸纤维膜或是尼龙膜上。DNA是通过它们的磷酸核糖骨架与膜结合，它们的碱基是自由的，可以和互补的核酸分子配对。,然后把标记好的探针加热变性，并与膜杂交。经过一段时间，等杂交完成后，洗去没有结合的探针，接着就可以检测结合到膜上的探针的位置了。,
