包含的cDNA克隆数目较少课件.ppt

1、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 Elrctrophoresis 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

2、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 ProteinPart 6 Part 6 基因文库和基因文库和cDN

3、AcDNA文库的构建文库的构建Construction of DNA&cDNA Library 基因组文库（genomic library）：包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）：包含细胞包含细胞的全部的全部mRNA的信息的的信息的重组cDNA克隆群体克隆群体称为称为cDNA库。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

4、是分子水平，而对特定基因片段的分离和获得是上述研究的基础。上述研究的基础。完整的基因组文库完整的基因组文库(genomic library)(genomic library)的构的构建使任何建使任何DNADNA片段的筛选和获得成为可能。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200-500kb）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

5、。(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。由于由于mRNA含有某种细胞的各种含有某种细胞的各种RNA分子，分子，因而反转录合成的因而反转录合成的cDNA将代表各样将代表各样mRNA拷拷贝，将其和载体贝，将其和载体DNA重组，并转化到宿主细菌重组，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。里或包装成噬菌体颗粒，得到一系列克隆群体。每个克隆只含一种每个克隆只含一种mRNA的信息，足够数目克的信息，足够数目克隆

6、的总和则包含细胞的全部隆的总和则包含细胞的全部mRNA的信息，这的信息，这样的克隆群体叫样的克隆群体叫cDNA库。库。2.1 概述二、cDNA文库（cDNA library）按照筛选方式不同按照筛选方式不同cDNA库可分为：库可分为：表达型表达型cDNA文文库库:采用表达型载体。插入的采用表达型载体。插入的cDNAcDNA片段可表达产生融合蛋白，不能采用核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产物发生特异性结合的抗体或化合物进行标记筛选。记筛选。非表达型非表达型cDNA文文库：适用于那

7、些采用核苷库：适用于那些采用核苷酸探针进行杂交筛选的基因。酸探针进行杂交筛选的基因。根据载体的不同将根据载体的不同将cDNAcDNA库分为：库分为：质粒质粒cDNAcDNA库库:包含的包含的cDNAcDNA克隆数目较少，适于克隆数目较少，适于较高丰度的较高丰度的mRNAmRNA噬菌体噬菌体cDNAcDNA库库:包含的包含的cDNAcDNA克隆数目非常多，克隆数目非常多，适用于那些低丰度和极低丰度的适用于那些低丰度和极低丰度的mRNAmRNA 在构建一个在构建一个cDNAcDNA库时，首先应考虑的问题是库时，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，可筛选方法。若采用核苷酸探针进

8、行杂交筛选，可构建表达型或非表达型构建表达型或非表达型cDNAcDNA库；如利用蛋白质的库；如利用蛋白质的生物活性或免疫原性进行筛选，则只能构建表达生物活性或免疫原性进行筛选，则只能构建表达型型cDNAcDNA库。库。其次应考虑的问题是其次应考虑的问题是mRNAmRNA的丰度。对于高丰的丰度。对于高丰度的度的mRNAmRNA所需构建的所需构建的cDNAcDNA库相对较小；而极低丰库相对较小；而极低丰度的度的mRNAmRNA，如仅占，如仅占mRNAmRNA总量的总量的1/101/106 6的某些的某些mRNAmRNA，所需构建的所需构建的cDNAcDNA库则必须很大，尽可能包括其对库则必须很大，

9、尽可能包括其对应的克隆。应的克隆。2.2 2.2 构建构建cDNAcDNA库主要包括以下几个步骤：库主要包括以下几个步骤：mRNA mRNA的分离；的分离；cDNA cDNA第一链的合成；第一链的合成；cDNA cDNA第二条链的合成；第二条链的合成；cDNA cDNA与载体的连接；与载体的连接；噬菌体的包装及转染或质粒的转化。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文库构建的具体操作方法见有关实验指南。有关实验指南。UNIT 2MANIPULATION OF DNA AND GENE ISOLATIONLECTURES:9.DNA

10、 Cloning and Library Construction10.Isolating Genes9.DNA Cloning and Library Constructiona).DNA cloningi).Restriction endonucleasesii).Cloning vectorsiii).The process of cloning a segment of DNAb).Library constructioni).Genomic librariesii).cDNA libraries How does one isolate a gene for an inherited

11、 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 proteinRestriction endonucleases Restriction enzymes cut DNA into specific fragments Restriction enzymes re

12、cognize 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-modification systemBacteria have enzy

13、mes 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 genus,species,strain,and type5.Recog

14、nize 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 cutter46=4,096 bp 8-base cutter48

15、=65,536 bp4-base cutter:cuts DNA into 256 bp average-sized fragments in a random sequenceevery 256 bp:NO256 bp average-size fragments:YESBar=256 bpProducts generated by restriction enzymes COHESIVE ENDSEcoRI5GAATTC35GAATTC33CTTAAG53CTTAA G5PstI5CTGCAG35CTGCA G33GACGTC53GACGTC5 BLUNT ENDSHaeIII5GGCC3

16、 5GG CC33CCGG5 3CC GG5Formation of recombinant DNA moleculescut DNAsmix together fragments and anneal cohesive endsseal 3,5 ends by DNA ligaserecombinant DNAsVectors used in molecular cloningVector Insert (and host)Characteristics size rangePlasmid Small circular DNA 5-10 kb (bacteria,yeast)Bacterio

17、phage 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 replicationStructure of pBR322-a common cloning vector derived fr

18、om 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.coliEcoRISal I gene for ampicillinresistancegene for

19、tetracycline resistancePst IoriCloning 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 isolatedPstIHuman DNA cut with PstIPPpBR322 ampR,tetRpBR322(human clone)tetRPPampRtetRpBR322 DNA cut with P

20、stIinactivating the ampR genetetRtetRcombineandligateLibrary 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 bacteriophage vectorsConstruction of a genomic libraryvecto

21、r 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 sitesinternal fragment(dispensable for phage growth)NNG GATCCNN NNCCTAG GNNinternal fragmentc

22、ut with Bam HI(6-base cutter)remove internalfragment“left arm”“right arm”cut with Sau 3A(4-base cutter)which has ends compatible with Bam HI:NNN GATCNNN NNNCTAG NNNisolate 20 kb fragmentshuman genomic DNA(isolated from many cells)Bam HI sites:combine and treatwith DNA ligase“left arm”“right arm”“lef

23、t arm”“right arm”package into bacteriophage and infect E.coli123456 genomic library of human DNA fragments in which each phage contains a different human DNA sequence7 isolation of 20 kb fragments provides optimallysized DNAs for cloning in bacteriophage partial digestion with a frequent-cutter(4-ba

24、se 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 the sequence da

25、ta 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 fragmentsa“contig”All possible sites:Results of

26、a partial digestion:=uncut=cutConstruction of a cDNA library reverse transcriptase makes a DNA copy of an RNAThe life cycle of a retrovirus depends on reverse transcriptaseretrovirus1.virus enters celland looses envelope2.the capsid is uncoated,releasing genomicRNA and reverse transcriptase3.reverse

27、 transcriptase makes a DNA copy4.then copies the DNA strand to make it double-stranded DNA,removing the RNA with RNase H5.the DNA is then integratedinto the host cell genomewhere it is transcribed byhost RNA polymerase II6.it is translated into viral proteins,and assembled into newvirus particlesnew

28、 viruses cDNA library constructionAAAAA53 mRNA(all mRNAs in cell)anneal oligo(dT)primers of 12-18 bases in lengthAAAAATTTTT535add reverse transcriptase and dNTPsAAAAATTTTT5335 cDNAadd RNaseH(specific for the RNA strand of an RNA-DNA hybrid)and carry out a partial digestionAATTTTT5short RNA fragments

29、 serve as primers for second strand synthesis using DNA polymerase I 33AAAAATTTTT53short RNA fragments serve as primers for second strand synthesis using DNA polymerase I AAATTTTT53DNA polymerase I removes the remaining RNA with its 5 to 3 exonuclease activity and continues synthesisDNA ligase seals

30、 the gaps AAATTTTT53AAAAATTTTT53double-stranded cDNAAAAAATTTTT53NNNNNNNNGNNNNNNNNCTTAAEcoRI linkers are ligated to both endsusing DNA ligaseAAAAANNNNNNNNGTTTTTNNNNNNNNCTTAA double-stranded cDNA copies of mRNA with EcoRI cohesive ends are now ready to ligate into a bacteriophage lambda vector cut wit

31、h EcoRI53AATTCNNNNNNNN GNNNNNNNNEcoRI sitescombine cDNAs with lambda arms and treat with DNA ligase“left arm”“right arm”“left arm”“right arm”package into bacteriophage and infect E.coli123456 cDNA library in which each phage contains a different human cDNA7cDNAscDNA LibraryThe Central DogmaDNAmRNAPr

32、oteinSingle-strandedDouble-strandedPrecursor RNAexonintronAAAAAAAAAAnAAAAAAAAAAn Single-strandedAAAAAAAAAAndouble-strandedReverse transcriptioncDNAcDNAA cDNA or complementary DNA,is a DNA copy of an RNA,usually mRNAcDNAmRNADouble stranded Single stranded StableunstableEasy to manipulateMore difficul

33、t to manipulateNeed to be transcribed into RNA to make a protein Can be directly used to makea protein cDNA synthesisTTTTTTTTTFirst strandNick translationcDNA libraryEcoR IGenomic LibraryInfect cellsAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

34、AAAAAAAcDNA synthesisInfect cellscDNA libraryGenomic librarycDNA libraryGenomic DNAmRNASourceSpecies or strainsSpecies or strainsTissuesDevelopmental stagesVariation12k-20k0.2k-6kInsert sizeEqual Correlate with expression levelRepresentationOnly oneExpression vs.nonexpressionTypeDNADNA or antibody o

35、rproteinProbeGene structureInfer protein identityEncoded proteinInfer protein identityPurposeEcoR ISubcloning of the cDNA insert+EcoR IEcoR IIs there a better way to subclone the insert?amplifyPCR-polymerase chain reactionPCR is one of the most powerful molecular biology techniques.It allows scienti

36、sts 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 reactionTaq DNA polymeraseForward primerReverse primer5353535355333353555333355553The ideal PCR productsHeat1st cycleHeat2nd cycleHeat33535553333555

37、53353535533553533553353535355353353rd cycleThe ideal PCR products5335Heat4th cycle53355335533553355335Heat5th cycleHeat6th cycleHeatnth cycle53352n products5335533553355335533553355335533553355335533553355335Cloning out cDNA insert by PCRPCREcoR IEcoR I+EcoR IEcoR IEcoR I“I tried very hard but could

38、 not isolate the cDNA clonefrom 1 million cDNA clones”mRNA level is very very lowRT-PCRmRNAcDNA(cDNA)nTTTTT-Reverse Transcription+PCRYou need to know at least partial sequence of the cDNA you want to amplify!PCRRACE method to isolate full-length cDNA Rapid amplification of cDNA endsReverse transcrip

39、taseRNase HTerminal transferaseCCCCCOligo(dG)n pimerDNA polymeraseCCCCCGGGGGPCRLigate to TTTT Partial cDNAFull-length cDNACCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGCCCCCGGGGGmRNA is too longExpression of a cloned gene to study its protein functions1)Expression o

40、f a cloned mammalian gene in mammalian cellsA genomic clone(with introns)can be used.A cDNA(without introns)clone can be used 2)Expression of a cloned gene in a heterologous systema.in bacteriab.in yeastc.in insect cellsin each case,cDNA is preferred to be used,since youdont know whether a mammalian

41、 gene can be spliced correctly in a heterologous system.cDNAmRNAProteinBacterial protein expression vectors and systems-peptide orN-terminus of b b-galactosiaseMultiple cloning sitesG GAT CCC ATG AGG ACC CAT AGC AAT TCG GGG CCC CCT GGG AGG GCT Asp Pro Met Arg Thr His Ser Asn Ser Gly Pro Pro Gly Arg

42、AlaBamHIcDNAATG ACC ATG ATT ACG AAT TCG AGC TCG GTA CCC GGG GAT CCC ATG AGG ACC CAT AGC AAT TCG GGG CCC CCT GGG AGG GCTMet Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Met Arg Thr His Ser Asn Ser Gly Pro Pro Gly Arg AlaBam HIExpression vectorsTo yield the product of a cloned gene for further

43、studies1)Expression vectors with a strong promoterMore mRNA More proteinExpression vectors with a strong promoterExpression vectorsTo yield the product of a cloned gene for further studies1)Expression vectors with a strong promoterMore mRNA More protein2)Expression vectors with an inducible promoter

44、Foreign proteins when overexpressed could be toxicKeep the gene expression off till it is time to turn it ona.Drug-inducible(e.g.IPTG or arabinose)b.Heat-inducibleExpression vectorsTo yield the product of a cloned gene for further studies1)Expression vectors with a strong promoterMore mRNA More prot

45、ein2)Expression vectors with an inducible promoterForeign proteins when overexpressed could be toxicKeep the gene expression off till it is time to turn it ona.Drug-inducible(e.g.IPTG or arabinose)b.Heat-inducible3)Expression vectors with a fusion tag for affinity purificationFacilitate the purification of the expressed protein1)6 Histidine tag2)Glutathione transferase tag(GST)3)Maltose-binding protein tag