1、四、DNA克隆,1.克隆载体2.cDNA克隆和分析3.基因组克隆和分析,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,A vector should have four characteristics:Ability to replicate independently of the host cellA recognition sequence for a restriction enzymeA reporter geneSma
2、ll size in comparison with hosts chromosomes,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin 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 vectorse.g., bacteriophage. The
3、 genes that cause host cell to lyse can be cut out and replaced with other DNA.Bacterial plasmids dont work for yeasts because the origins of replication use different sequences.A yeast artificial chromosome (YAC) has been created: contains yeast origin of replication, plus yeast centromere and telo
4、mere sequences.Also contains artificial restriction sites and reporter genes,FACTS,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,1.克隆载体,概论质粒噬菌体载体病毒载体 SV40 反转录病毒 腺病毒 AAV 慢病毒酵母载体（包括Yep,Ycp,Yip,YAC),Dept. of Biochemistry and Molecular Biology,
5、 School of Basic Medical Sciences, Tianjin Medical University,质粒, plasmid,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 replication or o
6、ri), 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 of the plasmid in cells, is usually dominant and is usually a gene encoding resistance to some antibiotic.
7、The cloning site is a restriction endonuclease cleavage site into which foreign DNA can be inserted without interfering with the plasmids ability to replicate or to confer the selectable phenotype on its host.,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Med
8、ical University,ColE1 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 upstream of the ori. Appropriate processing of the RNAII tran
9、script 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 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
10、form the primer is regulated by a second transcript, RNAI. RNAI is complementary 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 inhibited.
11、 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, Tianjin Medical University,Dept. of Biochemistry and Molecular Biology, School of Bas
12、ic Medical Sciences, Tianjin Medical University,A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species, such as E.coli and yeast.,,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,24
13、 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,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Ti
14、anjin Medical University,Human cytomegalovirus (CMV) immediate early promoter: 1589;Enhancer region: 59465; TATA box: 554560; Transcription start point: 583 MCS: 591665 IRES sequence: 6661250 Enhanced green fluorescent protein (EGFP) geneKozak consensus translation initiation site: 12471257Start cod
15、on (ATG): 12541256; Stop codon: 19711973Insertion of Val at position 2: 12571259GFPmut1 chromophore mutations (Phe-64 to Leu; Ser-65 to Thr): 14461451 His-231 to Leu mutation (AT): 1948Polyadenylation signals: 21272132 CA (Arg to Ser) mutation to remove BssH II site: 3732 Herpes simplex virus (HSV)
16、thymidine kinase (TK) polyadenylation signals: 42344252 pUC plasmid replication origin: 45835226,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,噬菌体载体，phagemid,Plasmids have been developed that contain a filamentous phage origin of replicatio
17、n 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 Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical U
18、niversity,病毒载体,SV40 反转录病毒 腺病毒 rAAV 慢病毒,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Example of the construction of a replication-defective genomic HSV-1 vector containing foreign gene sequences by homologous recombination. Foreign gene cas
19、settes can be introduced 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 parental virus and provides free ends, thereby increasing the frequency
20、 of recombination. The BamHI B plasmid construct in which the foreign gene of interest replaces the ICP27 gene sequences is cotransfected along with the ICP27 replication-defective mutant virus DNA into ICP4/ICP27-complementing 7B cells. Following homologous recombination, clear plaque isolates are
21、purified from blue plaque parental virus by limiting 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 promotor; ICP, infected cell protei
22、n; 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 interest is first cloned into a shuttle vector (e.g., pAdTrack-CMV). The resultant plasmid is linearized by digesting wit
23、h restriction endonuclease PmeI, and subsequently cotransformed into E. coli. BJ5183 cells with an adenoviral backbone plasmid (e.g., pAdEasy-1). Recombinants are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant
24、plasmid is transfected into adenovirus packaging cell lines (e.g., 293 cells). Recombinant adenoviruses are typically generated within 7 to 12 days. The “left arm” and “right arm” represent the regions mediating homologous recombination between the shuttle vector and the adenoviral backbone vector.
25、Abbreviations: An, polyadenylation site; Bm, BamHI, RI, EcoRI; LITR, left-hand inverted terminal repeat (ITR) and packaging signal; RITR, right-hand ITR; Sp, SpeI.,Retroviral vectors. Retroviral vectors that contain selectable drug markersneomycin phosphotransferase (neo), histidinol dehydrogenase (
26、hisD), or hygromycin phosphotransferase (hph)are shown with their 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 (LTR); N, neomycin (neo) g
27、ene; pA, polyadenylation signals; S, SV40 early promotor; X, cloning site; +, extended retroviral packaging signal.,rAAV vector,The production of adenovirus-free rAAV particles requires four elements: an rAAV vector plasmid containing the transgene anked by AAV inverted terminal repeats (ITRs)a plas
28、mid that supplies the AAV viral proteins necessary for replicating and packaging the rAAV sequences (AAV helper 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
29、and the cells are harvested. The rAAV is then puried 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 transfected using three plasmids: the plasmid carrying
30、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 plasmid supplying the adenovirus helper genes E2, E4, and VA RNA genes (pXX6, AD DNA), thereby generating Ad-free rAAV.,Iodixanol (碘海醇） ste
31、p gradient before and after centrifugation. The step gradient is generated by underlying 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 (cl
32、ear) 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,慢病毒载体，Lentiviral vector,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianj
33、in Medical University,Dept. of 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 Biochemistry and Molecular Biology, School of Basic Medic
34、al Sciences, Tianjin Medical University,载体的应用,Phagemid 产生单链DNA，早期用于测序基因克隆表达重组蛋白 基因治疗的载体产生融合蛋白（细胞中定位等）蛋白相互作用鉴定 （基因功能分析）等,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,体外in vitro 重组DNA的形成： DNA ligation,1. Sticky-end ligation2. Blunt-end ligat
35、ion3. 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 togethertwo (or more) inserts ligated togetherseveral DNAs ligated together, but not circularized1 vector
36、ligated to 1 insert DNA,DNA Ligation,ligating vector to insert,+,each cut with the same RE,DNA ligase,4300 bp; 100ng; 1.7 x 1011 molecules,900 bp; 63ng; 5.7 x 1010 molecules,Insert Vector 10x Ligase buffer uLT4 DNA ligase (10u/uL) uL H20 in the volume of 10-20uL, RT for several hours,Transformation
37、in E. coli,Transformation is a very inefficient process1g 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 制备的化学感受态E.coli细胞Electroporation: 低电导率的感受态E.coli细
38、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 types of plasmid DNA (lanes 7 to 11). Lanes: 1, lambda HindIII marker; 2, cleared lysate before column purification; 3, flowthrou
39、gh fraction; 4,5, first and second Buffer QC washes; 6, eluted plasmid DNA; 7, fraction containing 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 chromoso
40、mal DNA; 11, fraction 10 digested with EcoRI; 12, lambda HindIII marker.,Appropriate Agarose Concentrations for Separating DNA Fragments of Various Sizes,思考题：,产生DNA克隆的实验步骤有哪些？ 1.载体和目的DNA片段的获得（合成、PCR、酶切等）和连接2.转化到感受态细胞E.coli3.转化子/菌落/克隆的获得与鉴定（酶切、PCR、序列测定等）,2.cDNA克隆和分析,cDNA的合成cDNA文库的构建从DNA文库中筛选目标克隆选取cDN
41、A的分析5和/或3末端快速扩增技术,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,mRNA的提取,In 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
42、 rule, ve to ten major genes account for 20% of the 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). Since there are roughly 100,00
43、0 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 Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Preparation of Poly(A)
44、 + RNA,This protocol separates poly(A) + RNA from the remainder of total RNA, which is largely rRNA and tRNA. 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) +
45、 RNA is eluted by removing 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 Medical University,cDNA文库的构建,Dept. of Biochemistry and M
46、olecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Outline of cDNA synthesis and preparation for insertion into a vector.,Dept. of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University,Schematic outline of DSN-based cDNA normaliz
47、ation. The black and gray lines represent abundant and rare transcripts, respectively. 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 di
48、ffer between the 3and 5 adapters, respectively, thereby allowing directional cloning of the cDNA library.,Agarose gel electropherogram of non-normalized (lane 1) and normalized cDNA(lanes 2 to 4) that has been PCR amplied using a too-long extension step (6 min). No bright bands are visible, and the normalized cDNA appears as a smear starting from the high-molecular-weightregion of the gel. Lane M shows a 1-kb DNA ladder.,
