四dna克隆 ppt課件

1、四、DNA克隆,1.克隆載體2.cDNA克隆和分析3.基因組克隆和分析,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,A vector should have four characteristics:Ability to replicate independent

2、ly of the host cellA recognition sequence for a restriction enzymeA reporter geneSmall size in comparison with host’s chromosomes,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin

3、Medical University,Plasmids can be used for genes of 10,000 bp or less. Most eukaryote genes are larger than this.Viruses can be used as vectors—e.g., bacteriophage. The genes that cause host cell to lyse can be cut out

4、 and replaced with other DNA.Bacterial plasmids don’t work for yeasts because the origins of replication use different sequences.A yeast artificial chromosome (YAC) has been created: contains yeast origin of replicatio

5、n, plus yeast centromere and telomere sequences.Also contains artificial restriction sites and reporter genes,FACTS,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical Universi

6、ty,1.克隆載體,概論質(zhì)粒噬菌體載體病毒載體 SV40 反轉(zhuǎn)錄病毒 腺病毒 AAV 慢病毒酵母載體（包括Yep,Ycp,Yip,YAC),Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,質(zhì)粒, plasmi

7、d,All plasmid vectors contain three common features: a replicator, a selectable marker, and a cloning site.The replicator is a stretch of DNA that contains the site at which DNA replication begins (the origin of replic

8、ation or ori), and that also includes genes encoding whatever plasmid-encoded RNAs and proteins are necessary for replication. The selectable marker, necessary for following and maintaining the presence o

9、f the plasmid in cells, is usually dominant and is usually a gene encoding resistance to some antibiotic. The cloning site is a restriction endonuclease cleavage site into which foreign DNA can be inserted w

10、ithout interfering with the plasmid’s ability to replicate or to confer the selectable phenotype on its host.,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,ColE

11、1 replication initiation and copy number control. ColE1 replication requires the formation of a RNA primer to initiate DNA synthesis. The RNA primer is derived from processing of a transcript, RNAII (bold), that starts u

12、pstream of the ori. Appropriate processing of the RNAII transcript is dependent on formation of a persistent hybrid between RNAII and the ori DNA template.If the proper RNAII secondary structure forms, then the RNA/DNA

13、duplex is maintained at the ori and RNAse H can cleave the RNAII transcript to generate the primer for DNA synthesis. Processing of RNAII to form the primer is regulated by a second transcript, RNAI. RNAI is complementar

14、y to the 5′ end of RNAII. If the RNAI transcript forms a duplex with RNAII, RNAII cannot take on the secondary structure necessary to create the persistent hybrid at the ori and maturation of the RNAII primer is inhibite

15、d. The copy number of ColE1 plasmids is determined by the balance between successful RNAII processing events and those inhibited by RNAI.,,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Ti

16、anjin Medical University,,,,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate

17、in two different host species, such as E.coli and yeast.,,,www.promega.com,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,2

18、4 of 37 colonies contain vectors with inserts,8.5 x 107 cells are plated on IPTG/X-gal and antibiotics agar plate,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University

19、,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Human cytomegalovirus (CMV) immediate early promoter: 1–589;Enhancer region: 59–465; TATA box: 554–560; Transcrip

20、tion start point: 583? MCS: 591–665? IRES sequence: 666–1250? Enhanced green fluorescent protein (EGFP) geneKozak consensus translation initiation site: 1247–1257Start codon (ATG): 1254–1256; Stop codon: 1971–197

21、3Insertion of Val at position 2: 1257–1259GFPmut1 chromophore mutations (Phe-64 to Leu; Ser-65 to Thr): 1446–1451 His-231 to Leu mutation (A→T): 1948Polyadenylation signals: 2127–2132 & 2156–2161; mRNA 3' ends

22、: 2165 & 2177? f1 single-strand DNA origin: 2224–2679 (Packages the noncoding strand of EGFP.)? Bacterial promoter for expression of Kan r gene:–35 region: 2741–2746; –10 region: 2764–2769Transcription start p

23、oint: 2776? SV40 origin of replication: 3020–3155? SV40 early promoter/enhancer72-bp tandem repeats: 2853–2996; 21-bp repeats (3): 3000–3063Early promoter element: 3076–3082? Kanamycin/neomycin resistance gene: 3

24、204–3998G→A mutation to remove Pst I site: 3386; C→A (Arg to Ser) mutation to remove BssH II site: 3732? Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals: 4234–4252? pUC plasmid replication o

25、rigin: 4583–5226,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,噬菌體載體，phagemid,Plasmids have been developed that contain a filamentous phage origin of replic

26、ation in addition to a plasmid ori. These “phagemid” vectors can be grown and propagated as plasmids.,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,,,,Dept. of

27、 Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,病毒載體,SV40 反轉(zhuǎn)錄病毒 腺病毒 rAAV 慢病毒,Dept. of Biochemistry and Molecular Biology, School of Basic Medical S

28、ciences, Tianjin Medical University,Example of the construction of a replication-defective genomic HSV-1 vector containing foreign gene sequences by homologous recombination. Foreign gene cassettes can be intr

29、oduced into the ICP27? replication-defective mutant by homologous recombination following digestion of the mutant viral DNA with the PacI restriction endonuclease. PacI digestion reduces the background levels of parenta

30、l virus and provides free ends, thereby increasing the frequency of recombination. The BamHI B plasmid construct in which the foreign gene of interest replaces the ICP27 gene sequences is cotransfected along wi

31、th the ICP27? replication-defective mutant virus DNA into ICP4/ICP27-complementing 7B cells. Following homologous recombination, clear plaque isolates are purified from blue plaque parental virus by limi

32、ting dilution. RT-PCR analysis is employed to verify the expression of foreign gene. Abbreviations: BGH pA, bovine growth hormone polyadenylation and cleavage sequence; HCMV IEp, human cytomegalovirus immediate early

33、 promotor; ICP, infected cell protein; LAT, latency-associated transcript; UL, unique long segment of HSV-1 genome; US, unique short segment of HSV-1 genome.,Schematic overview of the AdEasy technology. The gene of

34、 interest is first cloned into a shuttle vector (e.g., pAdTrack-CMV). The resultant plasmid is linearized by digesting with restriction endonuclease PmeI, and subsequently cotransformed into E. coli. BJ5183 cells with an

35、 adenoviral backbone plasmid (e.g., pAdEasy-1). Recombinants are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfe

36、cted into adenovirus packaging cell lines (e.g., 293 cells). Recombinant adenoviruses are typically generated within 7 to 12 days. The “l(fā)eft arm” and “right arm” represent the regions mediating homologous recombination b

37、etween the shuttle vector and the adenoviral backbone vector. Abbreviations: An, polyadenylation site; Bm, BamHI, RI, EcoRI; LITR, left-hand inverted terminal repeat (ITR) and packaging signal; RITR, right-hand

38、 ITR; Sp, SpeI.,Retroviral vectors. Retroviral vectors that contain selectable drug markers—neomycin phosphotransferase (neo), histidinol dehydrogenase (hisD), or hygromycin phosphotransferase (hph)—are shown with t

39、heir GenBank accession numbers for the complete vector sequences. Abbreviations: C, cytomegalovirus (CMV) immediate early promotor; H, hygromycin-B-phosphotransferase (hph); HD, hisD gene; L, long terminal repeat

40、(LTR); N, neomycin (neo) gene; pA, polyadenylation signals; S, SV40 early promotor; X, cloning site; Ψ +, extended retroviral packaging signal.,rAAV vector,The production of adenovirus-free rAAV particles requires four e

41、lements: an rAAV vector plasmid containing the transgene ?anked by AAV inverted terminal repeats (ITRs)a plasmid that supplies the AAV viral proteins necessary for replicating and packaging the rAAV sequences (AAV help

42、er plasmid)a plasmid supplying the adenoviral helper genes (Ad helper plasmid), and tissue culture cells. After cotransfection of the plasmids into the tissue culture cells, rAAV is produced and the cells are harveste

43、d. The rAAV is then puri?ed either on CsCl gradients or on heparin-Sepharose columns and dialyzed for storage.,Generation of adenovirus-free recombinant adeno-associated virus. 293 cells(which supply the Ad E1 gene) are

44、 transfected using three plasmids: the plasmid carrying the transgene (sub201-gene “X”, AAV vector), the plasmid supplying the replication and capsid genes of AAV2 without terminal repeats (pXX2, AAV helper), and the pla

45、smid supplying the adenovirus helper genes E2, E4, and VA RNA genes (pXX6, AD DNA), thereby generating Ad-free rAAV.,Iodixanol (碘海醇） step gradient before and after centrifugation. The step gradient is generated by underl

46、ying the virus concentrate with 15%, 25%, 40%, and 60% iodixanol solutions. Percentages refer to the percent of iodixanol in each solution. After centrifugation, the virus resides exclusively in the 40% iodixanol (clear)

47、 layer. Care should be taken when removing this layer not to remove any of the 25% layer as this contains cell debris.,Preparation of Vector Mixes for Transient Transfection of 293T Cells,慢病毒載體，Lentiviral vector,Dept. of

48、 Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Dept. of Biochem

49、istry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,載體的應(yīng)用,Phagemid 產(chǎn)生單鏈DNA，早

50、期用于測序基因克隆表達重組蛋白 基因治療的載體產(chǎn)生融合蛋白（細胞中定位等）蛋白相互作用鑒定 （基因功能分析）等,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,體外in vitro 重組DNA的形成： DNA ligation,1. Sticky-end ligat

51、ion2. Blunt-end ligation3. Sticky-blunt-end ligation,ligation of vector to insert produces several productsvector ligated to itself (recircularized)insert ligated to itself (circularized, no ori)two vectors ligated

52、togethertwo (or more) inserts ligated togetherseveral DNAs ligated together, but not circularized1 vector ligated to 1 insert DNA,,DNA Ligation,ligating vector to insert,,,,,+,,each cut with the same RE,DNA ligase,~

53、4300 bp; 100ng; 1.7 x 1011 molecules,900 bp; 63ng; 5.7 x 1010 molecules,Insert Vector 10x Ligase buffer

54、uLT4 DNA ligase (10u/uL) uL H20 in the volume of 10-20uL, RT for several hours,,Transformation in E. coli,Transformation is a very inefficie

55、nt process1µg typical plasmid vector = 3 x 1011 copiesadded to highly competent E. coli cells yields at best109 antibiotic resistant colonies109/ 3 x 1011 = 1/300 vectors/transformed E. coli,Heat shock：CaCl2 制備的

56、化學(xué)感受態(tài)E.coli細胞Electroporation: 低電導(dǎo)率的感受態(tài)E.coli細胞,Agarose analytical gel (1%) comparing DNA composition of QIAGEN-tip elution fractions at different stages of plasmid purification (lanes 2 to 6) or containing different typ

57、es of plasmid DNA (lanes 7 to 11). Lanes: 1, lambda HindIII marker; 2, cleared lysate before column purification; 3, flowthrough fraction; 4,5, first and second Buffer QC washes; 6, eluted plasmid DNA; 7, fraction cont

58、aining denatured supercoiled DNA; 8, fraction containing multimeric forms of supercoiled plasmid DNA; 9, fraction containing linear plasmid DNA (pTZ19/EcoRI); 10, fraction contaminated with bacterial chromosomal

59、 DNA; 11, fraction 10 digested with EcoRI; 12, lambda HindIII marker.,Appropriate Agarose Concentrations for Separating DNA Fragments of Various Sizes,思考題：,產(chǎn)生DNA克隆的實驗步驟有哪些？ 1.載體和目的DNA片段的獲得（合成、PCR、酶切等）和連接2.轉(zhuǎn)化到感受態(tài)細胞E.co

60、li3.轉(zhuǎn)化子/菌落/克隆的獲得與鑒定（酶切、PCR、序列測定等）,2.cDNA克隆和分析,cDNA的合成cDNA文庫的構(gòu)建從DNA文庫中篩選目標(biāo)克隆選取cDNA的分析5’和/或3’末端快速擴增技術(shù),Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,mRNA的提取,I

61、n eukaryotic cells, mRNA constitutes ～ 1% to 5% of the total RNA, and the quantity of different transcripts varies across a range of thousands to tens of thousands. As a rule, ?ve to ten major genes account for 20% of th

62、e cellular mRNA expressed in eukaryotic cells, 500 to 2000 intermediate genes account for 40% to 60%, and the remaining 20% to 40% are composed of transcripts encoding 10,000 to 20,000 rare genes (Alberts et al., 1994).

63、Since there are roughly 100,000 to 300,000 mRNA molecules per cell, there are 20,000 to 60,000 molecules/cell of the most abundant RNAs and as few as 1 mRNA molecule/cell of the least abundant species.,Dept. of Biochemis

64、try and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Preparation of Poly(A) + RNA,This protocol separates poly(A) + RNA from the remainder of total RNA, which is largely rRNA and tRNA

65、. Total RNA is denatured to expose the poly(A) (polyadenylated) tails. Poly(A)-containing RNA is then bound to oligo(dT) cellulose, with the remainder of the RNA washing through. The poly(A) + RNA is eluted by removing

66、salt from the solution, thus destabilizing the dT:rA hybrid. The column can then be repeated to remove contaminating poly(A)? RNA.,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Me

67、dical University,cDNA文庫的構(gòu)建,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Outline of cDNA synthesis and preparation for insertion into a vector.,Dept. of Biochem

68、istry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Schematic outline of DSN-based cDNA normalization. The black and gray lines represent abundant and rare transcripts, respectively

69、. The rectangles represent the adapter sequences and their complements. Within the rectangles, white indicates the common external parts of the adapters, while gray and black correspond to the internal parts that differ