《包含的cDNA克隆数目较少课件.ppt》由会员分享，可在线阅读，更多相关《包含的cDNA克隆数目较少课件.ppt（63页珍藏版）》请在三一办公上搜索。
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 Elrctrophoresis 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,Part 6 基因文库和cDNA文库的构建Construction of DNA&cDNA Library,基因组文库（genomic library）：包含某种生物体全部基因的随机片段的重组DNA克隆群体称为基因组文库。cDNA文库（cDNA library）：包含细
3、胞的全部mRNA的信息的重组cDNA克隆群体称为cDNA库。,一、基因组文库（genomic library）,基因工程技术的迅速发展使人们对生物体基因的结构、功能、表达及其调控的研究深入到分子水平，而对特定基因片段的分离和获得是上述研究的基础。完整的基因组文库(genomic library)的构建使任何DNA片段的筛选和获得成为可能。,1.1 构建真核细胞基因组文库的载体：噬菌体（可插入基因片断约20kb）粘性质粒（可插入基因片断约46kb）酵母人工染色体克隆系统(yeast artificial chromosome cloning system),简称YACS。（可插入基因片断约200
4、-500kb）,1.2 应用入噬菌体构建基因组文库的基本步骤,(1)准备载体DNA。(2)提取高分子量真核细胞DNA：并选择合适的限制性内切酶进行部分降解。(3)分离大小合适的真核DNA片段。,(4)载体DNA与外源DNA连接。(5)连接产物在体外进行包装。(6)检测重组噬菌体的滴度，扩增、分装保存。,由于mRNA含有某种细胞的各种RNA分子，因而反转录合成的cDNA将代表各样mRNA拷贝，将其和载体DNA重组，并转化到宿主细菌里或包装成噬菌体颗粒，得到一系列克隆群体。每个克隆只含一种mRNA的信息，足够数目克隆的总和则包含细胞的全部mRNA的信息，这样的克隆群体叫cDNA库。,2.1 概述,
5、二、cDNA文库（cDNA library）,按照筛选方式不同cDNA库可分为：表达型cDNA文库:采用表达型载体。插入的cDNA片段可表达产生融合蛋白，不能采用核苷酸探针筛选的目的基因，可采用能与表达产物发生特异性结合的抗体或化合物进行标记筛选。非表达型cDNA文库：适用于那些采用核苷酸探针进行杂交筛选的基因。,根据载体的不同将cDNA库分为：质粒cDNA库:包含的cDNA克隆数目较少，适于较高丰度的mRNA噬菌体cDNA库:包含的cDNA克隆数目非常多，适用于那些低丰度和极低丰度的mRNA,在构建一个cDNA库时，首先应考虑的问题是筛选方法。若采用核苷酸探针进行杂交筛选，可构建表达型或非表
6、达型cDNA库；如利用蛋白质的生物活性或免疫原性进行筛选，则只能构建表达型cDNA库。其次应考虑的问题是mRNA的丰度。对于高丰度的mRNA所需构建的cDNA库相对较小；而极低丰度的mRNA，如仅占mRNA总量的1/106的某些mRNA，所需构建的cDNA库则必须很大，尽可能包括其对应的克隆。,2.2 构建cDNA库主要包括以下几个步骤：mRNA的分离；cDNA第一链的合成；cDNA第二条链的合成；cDNA与载体的连接；噬菌体的包装及转染或质粒的转化。检测重组噬菌体的滴度，扩增、分装保存。,cDNA文库构建的具体操作方法见有关实验指南。,UNIT 2MANIPULATION OF DNA AN
7、D GENE ISOLATIONLECTURES:9.DNA Cloning and Library Construction10.Isolating Genes,9.DNA Cloning and Library Construction,a).DNA cloningi).Restriction endonucleasesii).Cloning vectorsiii).The process of cloning a segment of DNAb).Library constructioni).Genomic librariesii).cDNA libraries,How does one
8、 isolate a gene for an inherited disorder?There are three options:Start with a candidate proteinDNA protein Start with a candidate mRNADNAmRNA Direct positional cloningDNAAll three options require the cloning of DNA.,DNA mRNA protein,Restriction endonucleases Restriction enzymes cut DNA into specifi
9、c fragments Restriction enzymes recognize specific base sequences in double-strandedDNA and cleave both strands of the duplex at specific places Characteristics of restriction enzymes:1.Cut DNA sequence-specifically2.Bacterial enzymes;hundreds are purified and available commercially3.Restriction-mod
10、ification systemBacteria have enzymes that will cleave foreign DNA;hence,“restrict”the entry of viralDNA.To prevent the bacterias own DNA from being cut,there is a second enzyme thatmethylates the same sites recognized by the restriction enzyme(modifies that site).4.Named(e.g.,EcoRI)for bacterial ge
11、nus,species,strain,and type5.Recognize specific 4-8 bp sequences sequences have symmetry(they are palindromes)after cutting the DNA,the cut ends are either blunt staggered(overhangs)-cohesive ends facilitate cloning the DNA6.Frequency of cutting 4-base cutter44=256 bp 5-base cutter45=1,024 bp 6-base
12、 cutter46=4,096 bp 8-base cutter48=65,536 bp,4-base cutter:cuts DNA into 256 bp average-sized fragments in a random sequence,every 256 bp:NO256 bp average-size fragments:YES,Bar=256 bp,Products generated by restriction enzymes,COHESIVE ENDSEcoRI5GAATTC35GAATTC33CTTAAG53CTTAA G5PstI5CTGCAG35CTGCA G33
13、GACGTC53GACGTC5 BLUNT ENDSHaeIII5GGCC3 5GG CC33CCGG5 3CC GG5,Formation of recombinant DNA molecules,cut DNAs,mix together fragments and anneal cohesive ends,seal 3,5 ends by DNA ligase,recombinant DNAs,Vectors used in molecular cloningVector Insert(and host)Characteristics size rangePlasmid Small ci
14、rcular DNA 5-10 kb(bacteria,yeast)Bacteriophage lambda Linear viral DNA up to 20 kbor phage lambda(bacteria)Cosmid Hybrid of plasmid up to 50 kb(bacteria)and phageYeast artificial DNA containing yeast200 to 1000 kbchromosome or YAC centromere,telomeres,(yeast)and origins of replication,Structure of
15、pBR322-a common cloning vector derived from a naturally occurring plasmid has antibiotic resistance genes for selection of transformants containing the plasmid has unique restriction enzyme cleavage sites forinsertion of foreign DNA has origin of DNA replication(ori)for propagation in E.coli,EcoRI,S
16、al I,gene for ampicillinresistance,gene for tetracycline resistance,Pst I,Cloning a segment of DNA into a plasmid vector,bacteria are“transformed”with the recombinant plasmid colonies that grow in tetracycline,but not in ampicillin are isolated,PstI,Human DNA cut with PstI,P,P,pBR322 ampR,tetR,pBR32
17、2(human clone)tetR,P,P,ampR,tetR,pBR322 DNA cut with PstIinactivating the ampR gene,tetR,tetR,combineandligate,Library construction two types of libraries a genomic library contains fragments of genomic DNA(genes)a cDNA library contains DNA copies of cellular mRNAs both types are usually cloned in b
18、acteriophage vectorsConstruction of a genomic library,vector DNA(bacteriophage lambda)lambda has a linear double-stranded DNA genome the left and right arms are essentialfor the phage replication cycle the internal fragment is dispensable,“left arm”,“right arm”,Bam HI sites,internal fragment(dispens
19、able for phage growth),NNG GATCCNN NNCCTAG GNN,internal fragment,cut with Bam HI(6-base cutter),remove internalfragment,cut with Sau 3A(4-base cutter)which has ends compatible with Bam HI:NNN GATCNNN NNNCTAG NNN,isolate 20 kb fragments,human genomic DNA(isolated from many cells),Bam HI sites:,combin
20、e and treatwith DNA ligase,package into bacteriophage and infect E.coli,1,2,3,4,5,6,genomic library of human DNA fragments in which each phage contains a different human DNA sequence,isolation of 20 kb fragments provides optimallysized DNAs for cloning in bacteriophage partial digestion with a frequ
21、ent-cutter(4-base cutter)allows productionof overlapping fragments,since not every site is cut overlapping fragments insures that all sequences in the genome are cloned overlapping fragments allows larger physical maps to be constructed ascontiguous chromosomal regions(contigs)are put together from
22、the sequence data number of clones needed to fully represent the human genome(3 X 109 bp)assuming 20 kb fragments theoretical minimum=150,000 99%probability that every sequence is represented=800,000,Partial restriction enzyme digestionallows cloning of overlapping fragments,a“contig”,All possible s
23、ites:,Results of a partial digestion:,=uncut,=cut,Construction of a cDNA library reverse transcriptase makes a DNA copy of an RNA,The life cycle of a retrovirus depends on reverse transcriptase,retrovirus,1.virus enters celland looses envelope,2.the capsid is uncoated,releasing genomicRNA and revers
24、e transcriptase,3.reverse transcriptase makes a DNA copy,4.then copies the DNA strand to make it double-stranded DNA,removing the RNA with RNase H,5.the DNA is then integratedinto the host cell genomewhere it is transcribed byhost RNA polymerase II,6.it is translated into viral proteins,and assemble
25、d into newvirus particles,new viruses,cDNA library construction,AAAAA,5,3 mRNA(all mRNAs in cell),anneal oligo(dT)primers of 12-18 bases in length,AAAAATTTTT,5,35,add reverse transcriptase and dNTPs,AAAAATTTTT,53,35 cDNA,add RNaseH(specific for the RNA strand of an RNA-DNA hybrid)and carry out a par
26、tial digestion,AATTTTT,5,short RNA fragments serve as primers for second strand synthesis using DNA polymerase I,3,3,AAAAATTTTT,53,short RNA fragments serve as primers for second strand synthesis using DNA polymerase I,DNA ligase seals the gaps,AAAAATTTTT,53,double-stranded cDNA,AAAAATTTTT,53,NNNNNN
27、NNGNNNNNNNNCTTAA,EcoRI linkers are ligated to both endsusing DNA ligase,AAAAANNNNNNNNGTTTTTNNNNNNNNCTTAA,double-stranded cDNA copies of mRNA with EcoRI cohesive ends are now ready to ligate into a bacteriophage lambda vector cut with EcoRI,53,AATTCNNNNNNNN GNNNNNNNN,EcoRI sites,combine cDNAs with la
28、mbda arms and treat with DNA ligase,package into bacteriophage and infect E.coli,1,2,3,4,5,6,cDNA library in which each phage contains a different human cDNA,cDNAs,cDNA Library,The Central Dogma,DNA,mRNA,Protein,Single-stranded,Double-stranded,Precursor RNA,cDNA,cDNA,A cDNA or complementary DNA,is a
29、 DNA copy of an RNA,usually mRNA,cDNA synthesis,Nick translation,cDNA library,Genomic library,cDNA library,Subcloning of the cDNA insert,Is there a better way to subclone the insert?,amplify,PCR-polymerase chain reaction,PCR is one of the most powerful molecular biology techniques.It allows scientis
30、ts to amplify a specific DNA region in the test tube from extremely tiny amount of DNA sample(even from a single molecule of DNA).,PCR-polymerase chain reaction,Taq DNA polymerase,3rd cycle,Cloning out cDNA insert by PCR,+,“I tried very hard but could not isolate the cDNA clonefrom 1 million cDNA cl
31、ones”,mRNA level is very very low,RT-PCR,mRNA,cDNA,(cDNA)n,TTTTT,-Reverse Transcription+PCR,You need to know at least partial sequence of the cDNA you want to amplify!,RACE method to isolate full-length cDNA Rapid amplification of cDNA ends,GGGGG,Full-length cDNA,mRNA is too long,Expression of a clo
32、ned gene to study its protein functions,Expression of a cloned mammalian gene in mammalian cellsA genomic clone(with introns)can be used.A cDNA(without introns)clone can be used Expression of a cloned gene in a heterologous systema.in bacteriab.in yeastc.in insect cellsin each case,cDNA is preferred
33、 to be used,since youdont know whether a mammalian gene can be spliced correctly in a heterologous system.,cDNA,mRNA,Protein,Bacterial protein expression vectors and systems,Expression vectors,To yield the product of a cloned gene for further studies,Expression vectors with a strong promoter,More mR
34、NA,More protein,Expression vectors with a strong promoter,Expression vectors,To yield the product of a cloned gene for further studies,Expression vectors with a strong promoter,More mRNA,More protein,Expression vectors with an inducible promoterForeign proteins when overexpressed could be toxicKeep
35、the gene expression off till it is time to turn it on,Drug-inducible(e.g.IPTG or arabinose)Heat-inducible,Expression vectors,To yield the product of a cloned gene for further studies,Expression vectors with a strong promoter,More mRNA,More protein,Expression vectors with an inducible promoterForeign
36、 proteins when overexpressed could be toxicKeep the gene expression off till it is time to turn it on,Drug-inducible(e.g.IPTG or arabinose)Heat-inducible,3)Expression vectors with a fusion tag for affinity purificationFacilitate the purification of the expressed protein,6 Histidine tagGlutathione transferase tag(GST)Maltose-binding protein tag,
