新克隆技术不用酶切位点直接克隆

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,#,7/20/2019,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,#,7/20/2019,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,7/20/2019,#,In-Fusion,克隆技术介绍,September 22, 2024,主要内容,9/22/2024,2,1,In-Fusion,技术原理,3,In-Fusion,应用实例,4,In-Fusion,应用文献,2,In-Fusion,实验方法,In-Fusion,技术原理示意图,9/22/2024,3,In-Fusion,酶,Clontech,专利,50,，15 min,单管反应,In-Fusion,技术特点,9/22/2024,4,开放性,无缝,克隆,多片段,克隆,In-Fusion,不受载体限制,不受酶切位点限制,不附加任何冗余序列,一次反应,单管操作,阳性率高达,80%,主要内容,9/22/2024,5,1,In-Fusion,技术原理,3,In-Fusion,应用实例,4,In-Fusion,应用文献,2,In-Fusion,实验方法,实验方法,9/22/2024,6,引物设计,PCR,扩增,片段和载体酶切处理,连接体系建立,引物设计,PCR,扩增,载体线性化,连接体系建立,传统基因克隆操作流程,In-Fusion,基因克隆操作流程,引物设计,9/22/2024,7,普通,PCR,引物,In-Fusion,PCR,引物,保护碱基序列,完整的酶切位点,与目的基因片段相同,/,相互补,的特异碱基序列,15 bp,同源,碱,基序列,补齐酶切位点,缺失碱基,与目的基因片段相同,/,相互补,的特异碱基序列,5-,-3,-3,5-,In-Fusion,引物设计原则,9/22/2024,8,平末端和黏性末端均可直接进行,in-fusion,连接,选择与,载体末端相同,的,15 bp,同源碱基序列,补齐酶切位点缺失碱基,同源序列部分是与线性化载体末端,5end,前,15 bp,相互补的碱基序列,无需进行末端补平或者去磷酸化处理,若保留酶切位点，需补齐酶切位点缺失碱基,In-Fusion,引物设计原则,9/22/2024,9,平末端和黏性末端均可直接进行,in-fusion,连接,无需进行末端补平或者去磷酸化处理,In-Fusion,引物设计,-,原则,1,9/22/2024,10,黏性末端,（,5,悬挂）,黏性末端,（,3,悬挂）,平末端,In-Fusion,引物设计原则,9/22/2024,11,选择与,载体末端相同,的,15 bp,同源碱基序列,同源序列部分是与线性化载体末端,5end,前,15 bp,相互补的碱基序列,In-Fusion,引物,设计,-,原则,2,9/22/2024,12,黏性末端,（,5,悬挂）,黏性末端,（,3,悬挂）,平末端,In-Fusion,引物设计原则,9/22/2024,13,补齐酶切位点缺失碱基,若保留酶切位点，需补齐酶切位点缺失碱基,In-Fusion,引物,设计,-,原则,3,9/22/2024,14,补齐酶切位点,部分,缺失碱,基,In-Fusion,引物设计在线工具,9/22/2024,15,在线工具网址,片段与载体用量计算工具,PCR,引物转换工具,载体线性化,9/22/2024,16,PCR,扩增,酶切处理,建立,In-Fusion,连接体系,9/22/2024,17,Rxn Component,Cloning Rxn,5X In-Fusion HD Enzyme Premix 2 l,Linearized Vector x,l*,Purified PCR Fragment x,l*,dH,2,O x l,Total Volume 10,l,Linearized vector : Purified PCR fragment(,摩尔比,)=,1:2,*10 kb: 50-200 ng,*10 kb: 50-200 ng,主要内容,9/22/2024,18,3,In-Fusion,应用实例,1,In-Fusion,技术原理,4,In-Fusion,应用文献,2,In-Fusion,实验方法,In-Fusion,应用,9/22/2024,19,应用,多片段克隆,插入突变,构建载体模型,高通量克隆,In-Fusion,应用实例,9/22/2024,20,1,多片段克隆实验,2,插入突变实验,In-Fusion,TM,assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations.,Baogong Zhu, Guifang Cai, et al.,BioTechniques, Sep 2007;,43:354-359,In-Fusion,应用实例,-,多片段克隆,9/22/2024,21,多片段连接,-,构建真正无缝融合蛋白,将白细胞介素,2 (IL-2),分泌信号序列与,CD101,胞外域序列（删除内源性信号序列）和鼠免疫球蛋白,G3 (IgG3),的可结晶片段进行融合，构建无缝融合蛋白,IL-2 signal-CD101-Fc,，并将其转染,COS,细胞检测表达情况。,实验流程,设计引物；,扩增目的片段及载体线性化；,建立,In-Fusion,连接反应体系,。,In-Fusion,应用实例,-,多片段克隆,9/22/2024,22,引物设计并扩增目的片段,IL-2 Signal, CD101,和,Murine IgG3,IL-2,Signal with Overlaps to NcoI-Digested Vector and CD101, 88-bp PCR Product,Sense (NcoI underlined),5-,TTCAAATCCA,CCATG,G,ATAGAATGCAATTGTTG-3,Antisense,5-,CTGTTACTTCTCTCTG,AGAATTCGTAACCAAAGCCAAAGACAAAGCAATCA-3,CD101, 2799-bp PCR Product,Sense,5-,CAGAGAGAAGTAACAG,TTCAGAAA-3,Antisense,5-,GGCCGAGGAGCAGAT,CCTGGAA-3,Murine IgG3,with Overlaps to CD101 and SalI-Digested Vector, 771-bp PCR Product,Sense,5-,ATCTGCTCCTCGGCC,CCTAGAATACCCAAGCCCAGTACC-3,Antisense (SalI underlined),5-,AGTAACGTTA,GTCGA,C,TCAGTGTCTTGTAAGACCCGAGGA,-3,In-Fusion,应用实例,-,多片段克隆,9/22/2024,23,建立,In-Fusion,连接反应体系（,10,l,）,IL-2 Signal,CD101 Domain,Murine IgG3 Fc Tail,Vector,Total Transformants,Error-Free/,Sequence (No.),88 bp, 1.6 ng,2799 bp, 49.3 ng,771 bp, 13.6 ng,11,365 bp, 100 ng,690,1/2,结论：克隆阳性率为,50%,。转染,COS,细胞后，融合蛋白,IL-2 signal-,CD101-Fc,表达情况良好。,In-Fusion,应用实例,9/22/2024,24,1,多片段克隆实验,2,插入突变实验,In-Fusion,应用实例,-,插入突变,9/22/2024,25,插入突变,-,轻松实现插入突变,实验流程,确定突变位点；,设计引物；,扩增目的片段及载体线性化；,建立,In-Fusion,连接反应体系。,In-Fusion,应用实例,-,插入突变,9/22/2024,26,策略一,5 Sense Primer,(Overlap with Vector Underlined, SpeI Site Bold),5-,GCTCGGATCC,ACTAG,TCCAGTGTG-3,Antisense Mutant Primer (Mutation Underlined),206-bp PCR Product,5-,GA,AGC,GGATGAGTACA,GACAGGGCAAAGTC-3,Sense Mutant Primer (Mutation Underlined),5-,TGTACTCATCC,GCT,TC,TCACAACAGCAACAGC-3,3 Antisense Primer (BspEI Site Bold),509-bp PCR product,5-,TGTGGCTTCC,TCCGG,AAGGGTGCTTGGGGTTA-3,引物设计并扩增目的片段,206-bp,和,509-bp,将,human TIM-4 (hTIM-4),第,45,位的色氨酸,（,W,或,Trp,）,突变为丙氨酸（,A,或,Ala,），构建,hTIM-4 W45A,突变体。,In-Fusion,应用实例,-,插入突变,9/22/2024,27,建立,In-Fusion,连接反应体系（,10,l,）,6375-bp fragment containing the vector and 3 end of hTIM-4,206-bp PCR Product,509-bp PCR product,24 ng,1.6 ng,3.8 ng,结论：成功构建了,5,个,TIM-4,突变体，经过测序验证，其克隆阳性率,分别 为,3/3,、,3/3,、,2/3,、,2/3,、,1/8,。,In-Fusion,应用实例,-,插入突变,9/22/2024,28,策略二,4609-bp PCR Product,Sense Mutant Primer (Mutation Underlined),5-,AGT,TGA,GCCCAAATC,TTCCGACAAAACTCACACA-3,Antisense Primer,5-,GATTTGGGC,TCA,ACT,TTCTTGTCCACCTTGGTGT-3,将人免疫球蛋白,G1,（,IgG1,）第,103,位的半胱氨酸（,C,或,Cys,）突变为丝氨酸（,S,或,Ser,），构建,IgG1 C103S,突变体。,引物设计并扩增目的片段,4609-bp,结论：经过测序验证，克隆阳性率为,33%,。,In-Fusion,连接反应体系（,10,l,）,4609-bp PCR Product,20 ng,主要内容,9/22/2024,29,4,In-Fusion,应用文献,3,In-Fusion,应用实例,1,In-Fusion,技术原理,2,In-Fusion,实验方法,In-Fusion,应用文献,9/22/2024,30,Comparative Epigenomic Analysis of Murine and Human Adipogenesis,Tarjei S. Mikkelsen, Zhao Xu,et al.,Cell,Oct 2010,; 143 :,156169,In-Fusion BioBrick assembly and re-engineering,Sean C. Sleight, Bryan A. Bartley, et al.,Nucleic Acids Res,., May 2010; 38: 2624 - 2636.,Lumen Thiol Oxidoreductase1, a Disulfide Bond-Forming Catalyst, Is Required for the Assembly of Photosystem II in Arabidopsis,Mohamed Karamoko, Sara Cline, Kevin Redding, et al.,PLANT CELL, Dec 2011; 23: 4462 - 4475.,Ehrlichia chaffeensis TRP120 Interacts with a Diverse Array of Eukaryotic Proteins Involved in Transcription, Signaling, and Cytoskeleton Organization,Tian Luo, Jeeba A. Kuriakose, Bing Zhu, et al.,Infect. Immun,., Nov 2011; 79: 4382 - 4391.,GMechanism of auxiliary -subunit-mediated membrane targeting of L-type (CaV1.2) channels,Kun Fang and Henry M. Colecraft.,J. Physiol., Sep 2011; 589: 4437 - 4455.,Crimean-Congo Hemorrhagic Fever Virus-Encoded Ovarian Tumor Protease Activity Is Dispensable for Virus RNA Polymerase Function,ric Bergeron, Csar G. Albario, et al.,J. Virol,., Jan 2010; 84: 216 - 226.,CCS5, a Thioredoxin-like Protein Involved in the Assembly of Plastid c-Type Cytochromes,Stphane T. Gabilly, Beth Welty Dreyfuss, et al.,J. Biol. Chem., Sep 2010; 285: 29738 - 29749.,Major taste loss in carnivorous mammals,Peihua Jiang, Jesusa Josue, Xia Li, Dieter Glaser, et al.,PNAS, Mar 2012; 10.1073/pnas.1118360109.,技术支持,9/22/2024,31,：,800-810-6261,；,/,86,：,：,我们将竭诚为您服务！,9/22/2024,32,Thank You,！,3Q,！,