新克隆技術(shù)不用酶切位點(diǎn)直接克隆

1、In-Fusion克隆技術(shù)介紹September 30, 2022主要內(nèi)容9/30/20222 1In-Fusion技術(shù)原理 3In-Fusion應(yīng)用實(shí)例 4In-Fusion應(yīng)用文獻(xiàn) 2In-Fusion實(shí)驗(yàn)方法In-Fusion技術(shù)原理示意圖9/30/20223 In-Fusion酶Clontech專利50 ，15 min單管反應(yīng)In-Fusion技術(shù)特點(diǎn)9/30/20224 開放性無縫克隆多片段克隆In-Fusion不受載體限制不受酶切位點(diǎn)限制不附加任何冗余序列一次反應(yīng)單管操作陽性率高達(dá)80%主要內(nèi)容9/30/20225 1In-Fusion技術(shù)原理 3In-Fusion應(yīng)用實(shí)例 4In

2、-Fusion應(yīng)用文獻(xiàn) 2In-Fusion實(shí)驗(yàn)方法實(shí)驗(yàn)方法9/30/20226 引物設(shè)計(jì)PCR擴(kuò)增片段和載體酶切處理連接體系建立引物設(shè)計(jì)PCR擴(kuò)增載體線性化連接體系建立傳統(tǒng)基因克隆操作流程In-Fusion基因克隆操作流程引物設(shè)計(jì)9/30/20227 普通PCR引物In-Fusion PCR引物保護(hù)堿基序列完整的酶切位點(diǎn)與目的基因片段相同/相互補(bǔ)的特異堿基序列15 bp同源堿基序列補(bǔ)齊酶切位點(diǎn)缺失堿基 與目的基因片段相同/相互補(bǔ)的特異堿基序列5-3-35-In-Fusion引物設(shè)計(jì)原則9/30/20228 平末端和黏性末端均可直接進(jìn)行in-fusion連接選擇與載體末端相同的15 bp同源堿

3、基序列補(bǔ)齊酶切位點(diǎn)缺失堿基同源序列部分是與線性化載體末端5end前15 bp相互補(bǔ)的堿基序列無需進(jìn)行末端補(bǔ)平或者去磷酸化處理若保留酶切位點(diǎn)，需補(bǔ)齊酶切位點(diǎn)缺失堿基In-Fusion引物設(shè)計(jì)原則9/30/20229 平末端和黏性末端均可直接進(jìn)行in-fusion連接無需進(jìn)行末端補(bǔ)平或者去磷酸化處理In-Fusion引物設(shè)計(jì)-原則19/30/202210 黏性末端（5 懸掛）黏性末端（3懸掛） 平末端In-Fusion引物設(shè)計(jì)原則9/30/202211 選擇與載體末端相同的15 bp同源堿基序列同源序列部分是與線性化載體末端5end前15 bp相互補(bǔ)的堿基序列In-Fusion引物設(shè)計(jì)-原則29/

4、30/202212 黏性末端（5 懸掛）黏性末端（3懸掛） 平末端In-Fusion引物設(shè)計(jì)原則9/30/202213 補(bǔ)齊酶切位點(diǎn)缺失堿基若保留酶切位點(diǎn)，需補(bǔ)齊酶切位點(diǎn)缺失堿基In-Fusion引物設(shè)計(jì)-原則39/30/202214 補(bǔ)齊酶切位點(diǎn)部分缺失堿基In-Fusion引物設(shè)計(jì)在線工具9/30/202215 在線工具網(wǎng)址 片段與載體用量計(jì)算工具PCR引物轉(zhuǎn)換工具載體線性化9/30/202216 PCR擴(kuò)增酶切處理建立In-Fusion連接體系9/30/202217 Rxn ComponentCloning Rxn5X In-Fusion HD Enzyme Premix 2 lLine

5、arized Vector x l*Purified PCR Fragment x l*dH2O x lTotal Volume 10 lLinearized vector : Purified PCR fragment(摩爾比)=1:2*10 kb: 50-200 ng*10 kb: 50-200 ng主要內(nèi)容9/30/202218 3In-Fusion應(yīng)用實(shí)例 1In-Fusion技術(shù)原理 4In-Fusion應(yīng)用文獻(xiàn) 2In-Fusion實(shí)驗(yàn)方法In-Fusion應(yīng)用9/30/202219 應(yīng)用多片段克隆插入突變構(gòu)建載體模型高通量克隆In-Fusion應(yīng)用實(shí)例9/30/202220 1多

6、片段克隆實(shí)驗(yàn)2插入突變實(shí)驗(yàn) In-FusionTM assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Baogong Zhu, Guifang Cai, et al. BioTechniques, Sep 2007; 43:354-359In-Fusion應(yīng)用實(shí)例-多片段克隆9/30/202221 多片段連接-構(gòu)建真正無縫融合蛋白 將白細(xì)胞介素2 (IL-2)分泌信號(hào)序列與CD101胞外域序列（刪除內(nèi)源性信號(hào)序列）和鼠免疫球蛋白 G3 (IgG3)的可結(jié)晶

7、片段進(jìn)行融合，構(gòu)建無縫融合蛋白IL-2 signal-CD101-Fc ，并將其轉(zhuǎn)染COS細(xì)胞檢測(cè)表達(dá)情況。實(shí)驗(yàn)流程設(shè)計(jì)引物；擴(kuò)增目的片段及載體線性化；建立In-Fusion連接反應(yīng)體系。In-Fusion應(yīng)用實(shí)例-多片段克隆9/30/202222 引物設(shè)計(jì)并擴(kuò)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 ProductSense (NcoI underlined)5-TTCAAATCCACCATGGATAGAATGCAATTG

8、TTG-3Antisense5-CTGTTACTTCTCTCTGAGAATTCGTAACCAAAGCCAAAGACAAAGCAATCA-3CD101, 2799-bp PCR ProductSense5-CAGAGAGAAGTAACAGTTCAGAAA-3Antisense5-GGCCGAGGAGCAGATCCTGGAA-3Murine IgG3 with Overlaps to CD101 and SalI-Digested Vector, 771-bp PCR ProductSense5-ATCTGCTCCTCGGCCCCTAGAATACCCAAGCCCAGTACC-3Antisense

9、(SalI underlined)5-AGTAACGTTAGTCGACTCAGTGTCTTGTAAGACCCGAGGA-3In-Fusion應(yīng)用實(shí)例-多片段克隆9/30/202223 建立In-Fusion連接反應(yīng)體系（ 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結(jié)論：克隆陽性率為50%。

10、轉(zhuǎn)染COS 細(xì)胞后，融合蛋白IL-2 signal- CD101-Fc表達(dá)情況良好。In-Fusion應(yīng)用實(shí)例9/30/202224 1多片段克隆實(shí)驗(yàn)2插入突變實(shí)驗(yàn)In-Fusion應(yīng)用實(shí)例-插入突變9/30/202225 插入突變-輕松實(shí)現(xiàn)插入突變實(shí)驗(yàn)流程確定突變位點(diǎn)；設(shè)計(jì)引物；擴(kuò)增目的片段及載體線性化；建立In-Fusion連接反應(yīng)體系。In-Fusion應(yīng)用實(shí)例-插入突變9/30/202226 策略一5 Sense Primer (Overlap with Vector Underlined, SpeI Site Bold)5-GCTCGGATCCACTAGTCCAGTGTG-3Anti

11、sense Mutant Primer (Mutation Underlined)206-bp PCR Product5-GAAGCGGATGAGTACAGACAGGGCAAAGTC-3Sense Mutant Primer (Mutation Underlined)5-TGTACTCATCCGCTTCTCACAACAGCAACAGC-33 Antisense Primer (BspEI Site Bold)509-bp PCR product5-TGTGGCTTCCTCCGGAAGGGTGCTTGGGGTTA-3引物設(shè)計(jì)并擴(kuò)增目的片段206-bp 和509-bp 將human TIM-4 (

12、hTIM-4)第45位的色氨酸（W或Trp）突變?yōu)楸彼幔ˋ或Ala），構(gòu)建hTIM-4 W45A 突變體。In-Fusion應(yīng)用實(shí)例-插入突變9/30/202227 建立In-Fusion連接反應(yīng)體系（ 10 l ）6375-bp fragment containing the vector and 3 end of hTIM-4206-bp PCR Product509-bp PCR product24 ng 1.6 ng 3.8 ng 結(jié)論：成功構(gòu)建了5個(gè)TIM-4 突變體，經(jīng)過測(cè)序驗(yàn)證，其克隆陽性率 分別 為3/3、3/3、2/3、2/3、1/8。In-Fusion應(yīng)用實(shí)例-插入突變9

13、/30/202228 策略二4609-bp PCR ProductSense Mutant Primer (Mutation Underlined)5-AGTTGAGCCCAAATCTTCCGACAAAACTCACACA-3Antisense Primer5-GATTTGGGCTCAACTTTCTTGTCCACCTTGGTGT-3 將人免疫球蛋白G1（IgG1）第103位的半胱氨酸（C或Cys）突變?yōu)榻z氨酸（S或Ser），構(gòu)建IgG1 C103S突變體。引物設(shè)計(jì)并擴(kuò)增目的片段4609-bp結(jié)論：經(jīng)過測(cè)序驗(yàn)證，克隆陽性率為33%。In-Fusion連接反應(yīng)體系（ 10 l ）4609-bp PC

14、R Product20 ng 主要內(nèi)容9/30/202229 4In-Fusion應(yīng)用文獻(xiàn) 3In-Fusion應(yīng)用實(shí)例 1In-Fusion技術(shù)原理 2In-Fusion實(shí)驗(yàn)方法In-Fusion應(yīng)用文獻(xiàn)9/30/202230 Comparative Epigenomic Analysis of Murine and Human Adipogenesis Tarjei S. Mikkelsen, Zhao Xu, et al. Cell, Oct 2010 ; 143 : 156169In-Fusion BioBrick assembly and re-engineeringSean C.

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