无限制克隆技术研究进展及其应用
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摘要
无限制克隆(restriction-free cloning,RFC)是近年来建立起来的一种简单通用的DNA克隆技术,它可以精确地将目的片段插入到质粒内任意位置,是一种不受酶切位点、连接酶效率、目的基因长度或载体序列等条件限制的新型DNA重组技术.与其他多种克隆方法相比,RFC技术拥有不可替代的优势.本文在总结国内外RFC技术研究的基础上,系统阐述了RFC技术的原理、特点和在克隆方面的研究进展,并探讨其在分子生物学和合成生物学等领域的应用价值.
Restriction free cloning(RFC) is a simple and universal DNA cloning technology developed in recent years, which can accurately insert the target DNA sequence into any position of any plasmids. It is a novel DNA recombination method that is not limited by restriction endonuclease, efficiency of ligase, length of target DNA sequence or vector sequence. Compared with other cloning methods, RFC technology has irreplaceable advantages. On the basis of summarizing the researches of RFC at home and abroad, this manuscript systematically introduces the action principle, characteristics and research progress of RFC technology and discuss its application value in molecular biology and synthetic biology.
Restriction free cloning(RFC) is a simple and universal DNA cloning technology developed in recent years, which can accurately insert the target DNA sequence into any position of any plasmids. It is a novel DNA recombination method that is not limited by restriction endonuclease, efficiency of ligase, length of target DNA sequence or vector sequence. Compared with other cloning methods, RFC technology has irreplaceable advantages. On the basis of summarizing the researches of RFC at home and abroad, this manuscript systematically introduces the action principle, characteristics and research progress of RFC technology and discuss its application value in molecular biology and synthetic biology.
引文
[1]黄媛,陶颖,张文露,等.一种新的DNA分子克隆方法.中国科学:生命科学,2011,41(9):44-51Huang Y,Tao Y,Zhang W L,et al.Sci Chin Life Sci,2011,41(9):44-51
[2]Wilson R H,Morton S K,Deiderick H,et al.Engineered DNAligases with improved activities in vitro.Protein Engineering,Design&Selection:PEDS,2013,26(7):471-478
[3]Luft J R,Snell E H,Detitta G T.Lessons from high-throughput protein crystallization screening:10 years of practical experience.Expert Opinion On Drug Discovery,2011,6(5):465-480
[4]Geertsma E R,Poolman B.High-throughput cloning and expression in recalcitrant bacteria.Nat Methods,2007,4(9):705-707
[5]Liu Q,Li M Z,Leibham D,et al.The univector plasmid-fusion system,a method for rapid construction of recombinant DNAwithout restriction enzymes.Current Biology:CB,1998,8(24):1300-1309
[6]Reece-Hoyes J S,Walhout A J M.Gateway recombinational cloning.Cold Spring Harb Protoc,2018(1):1-6
[7]Park J,Throop A L,Labaer J.Site-specific recombinational cloning using gateway and in-fusion cloning schemes.Curr Protoc Mol Biol,2015,110(1):3.20.1-3.20.23
[8]韩占品,任健,于玮玮,等.花椰菜ERF114基因的克隆及Gateway技术构建表达载体.西南农业学报,2018,31(6):1128-1134Han Z P,Ren J,Yu W W,et al.Southwest China Journal of Agricultural Sciences,2018,31(6):1128-1134
[9]Li M Z,Elledge S J.MAGIC,an in vivo genetic method for the rapid construction of recombinant DNA molecules.Nat Genet,2005,37(3):311-319
[10]Garcíanafría J,Watson J F,Greger I H.IVA cloning:a single-tube universal cloning system exploiting bacterial in vivo assembly.Scientific Reports,2016,6(1):27459
[11]Wang Y,Liu Y,Chen J,et al.Restriction-ligation-free(RLF)cloning:a high-throughput cloning method by in vivo homologous recombination of PCR products.Genet Mol Res,2015,14(4):12306-12315
[12]Liu C J,Jiang H,Wu L,et al.OEPR cloning:an efficient and seamless cloning strategy for large-and multi-fragments.Scientific Reports,2017,7(1):44648
[13]Zhang Y,Werling U,Edelmann W.SLiCE:a novel bacterial cell extract-based DNA cloning method.Nucleic Acids Res,2012,40(8):e55
[14]Beyer H M,Gonschorek P,Samodelov S L,et al.AQUA cloning:a versatile and simple enzyme-free cloning approach.Plos One,2015,10(9):e0137652
[15]Li M Z,Elledge S J.SLIC:a method for sequence-and ligationindependent cloning.Methods in Molecular Biology,2012,852:51-59
[16]Jeong J Y,Yim H S,Ryu J Y,et al.One-step sequence-and ligationindependent cloning as a rapid and versatile cloning method for functional genomics studies.Applied&Environmental Microbiology,2012,78(15):5440-5443
[17]Yang J,Zhang Z,Zhang X A,et al.A ligation-independent cloning method using nicking DNA endonuclease.Biotechniques,2010,49(5):817-821
[18]Bitinaite J,Rubino M,Varma K H,et al.USER friendly DNAengineering and cloning method by uracil excision.Nucleic Acids Res,2007,35(6):1992-2002
[19]Geu-Flores F,Nour-Eldin H H,Nielsen M T,et al.USER fusion:a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products.NucleicAcids Res,2007,35(7):e55
[20]Nour-Eldin H H,Hansen B G,Norholm M H,et al.Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments.NucleicAcids Res,2006,34(18):e122
[21]Gibson D G,Young L,Chuang R Y,et al.Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods,2009,6(5):343-345
[22]Liu W,Ping Y,Tao W,et al.Homologous recombination-based DNA cloning methods and compositions:USA,US201213362382.2013-08-06.
[23]Sleight S C,Bartley B A,Lieviant J A,et al.In-Fusion BioBrick assembly and re-engineering.Nucleic Acids Res,2010,38(8):2624-2636
[24]Berrow N S,Alderton D,Owens R J.The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning.Methods Mol Biol,2009,498:75-90
[25]Zhu B,Cai G,Hall E O,et al.In-fusion assembly:seamless engineering of multidomain fusion proteins,modular vectors,and mutations.Biotechniques,2007,43(3):354-359
[26]Zeng F,Zang J,Zhang S,et al.AFEAP cloning:a precise and efficient method for large DNA sequence assembly.BMCBiotechnology,2017,17(1):81
[27]Liang J,Liu Z,Low X Z,et al.Twin-primer non-enzymatic DNAassembly:an efficient and accurate multi-part DNA assembly method.NucleicAcids Research,2017,45(11):e94
[28]Chen G J,Qiu N,Karrer C,et al.Restriction site-free insertion of PCR products directionally into vectors.Biotechniques,2000,28(3):498-500
[29]Van Den Ent F,Lowe J.RF cloning:a restriction-free method for inserting target genes into plasmids.Journal of Biochemical and Biophysical Methods,2006,67(1):67-74
[30]Zeng F,Hao Z,Li P,et al.A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends.BMC Biotechnology,2017,17(1):32
[31]Quan J,Tian J.Circular polymerase extension cloning of complex gene libraries and pathways.Plos One,2009,4(7):e6441
[32]Quan J,Tian J.Circular polymerase extension cloning for highthroughput cloning of complex and combinatorial DNA libraries.Nature Protocols,2011,6(2):242-251
[33]Bryksin A V,Matsumura I.Overlap extension PCR cloning:a simple and reliable way to create recombinant plasmids.Biotechniques,2010,48(6):463-465
[34]Jajesniak P,Wong T S.QuickStep-Cloning:a sequenceindependent,ligation-free method for rapid construction of recombinant plasmids.Journal of Biological Engineering,2015,9:15
[35]Qaidi S E,Hardwidge P R.ABC cloning:an efficient,simple,and rapid restriction/ligase-free method.MethodsX,2019,6:316-321
[36]Peleg Y,Unger T.Application of the Restriction-Free(RF)cloning for multicomponents assembly.Methods Mol Biol,2014,1116:73-87
[37]Unger T,Jacobovitch Y,Dantes A,et al.Applications of the restriction free(RF)cloning procedure for molecular manipulations and protein expression.Journal of Structural Biology,2010,172(1):34-44
[38]Bandyopadhyay B,Peleg Y.Facilitating circular permutation using restriction free(RF)cloning.Protein Engineering,Design&Selection:PEDS,2018,31(3):65-68
[39]Dai C,Miao C X,Lu G X.Site-directed mutagenesis based on overlap extension PCR.Progress in Modern Biomedicine,2010,10(03):177-192
[40]Guo Z Y,Hao X H,Yan J L,et al.A new method of site-directed mutagenesis by modifying overlap extension PCR.Biotechnology,2009,19(6):34-36
[41]Bond S R,Naus C C.RF-Cloning.org:an online tool for the design of restriction-free cloning projects.Nucleic Acids Research,2012,40(W1):W209-W13
[42]Steffens D L,Williams J G.Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.J Biomol Tech,2007,18(3):147-149
[43]Yang Y,Huang L,Wang J,et al.Expression,characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus.Enzyme and Microbial Technology,2015,68:43-49
[44]Tseng W C,Lin J W,Wei T Y,et al.A novel megaprimed and ligasefree,PCR-based,site-directed mutagenesis method.Analytical Biochemistry,2008,375(2):376-378
[45]Taniguchi N,Murakami H.Multiple site-directed and saturation mutagenesis by the patch cloning method.Methods in Molecular Biology,2017,1498:339-347
[46]Taniguchi N,Nakayama S,Kawakami T,et al.Patch cloning method for multiple site-directed and saturation mutagenesis.BMC Biotechnology,2013,13(1):1-8
[47]Zeng F,Zhang S,Hao Z,et al.Efficient strategy for introducing large and multiple changes in plasmid DNA.Sci Rep,2018,8(1):1714
[48]Erijman A,Dantes A,Bernheim R,et al.Transfer-PCR(TPCR):a highway for DNA cloning and protein engineering.Journal of Structural Biology,2011,175(2):171-177
[49]Steinmetz E J,Auldridge M E.Screening fusion tags for improved recombinant protein expression in E.coli with the Expresso?;solubility and expression screening system.Current Protocols in Protein Science,2017,5(27):1-20
[50]Berrow N S,Büssow K,Coutard B,et al.Recombinant protein expression and solubility screening in Escherichia coli:a comparative study.Acta Crystallographica Section D,Biological Crystallography,2010,62(10):1218-1226
[51]Perrakis A,Romier C.Assembly of protein complexes by coexpression in prokaryotic and eukaryotic hosts:an overview.Methods Mol Biol,2008,426:247-256
[52]Romier C,Ben Jelloul M,Albeck S,et al.Co-expression of protein complexes in prokaryotic and eukaryotic hosts:experimental procedures,database tracking and case studies.Acta crystallographica Section D,Biological Crystallography,2006,62(10):1232-1242
[53]陆路,熊文,张钰琨,等.RF克隆结合巢式PCR构建金黄色葡萄球菌组氨酸蛋白激酶AgrC表达载体.中国生物工程杂志,2014,34(10):55-60Lu L,Xiong W,Zhang Y K,et al.Journal of Chinese Biotechnology,2014,34(10):55-60
[54]Li M Z,Elledge S J.Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.Nat Methods,2007,4(3):251-256
[55]Hartley J L.Use of the gateway system for protein expression in multiple hosts.Current Protocols in Protein Science,2003,5(17):1-10
[56]Busso D,Delagoutte-Busso B,Moras D.Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli.Analytical Biochemistry,2005,343(2):313-321
[57]Moreland N,Ashton R,Baker H M,et al.A flexible and economical medium-throughput strategy for protein production and crystallization.Acta Crystallographica Section D,Biological Crystallography,2005,61(10):1378-1385
[2]Wilson R H,Morton S K,Deiderick H,et al.Engineered DNAligases with improved activities in vitro.Protein Engineering,Design&Selection:PEDS,2013,26(7):471-478
[3]Luft J R,Snell E H,Detitta G T.Lessons from high-throughput protein crystallization screening:10 years of practical experience.Expert Opinion On Drug Discovery,2011,6(5):465-480
[4]Geertsma E R,Poolman B.High-throughput cloning and expression in recalcitrant bacteria.Nat Methods,2007,4(9):705-707
[5]Liu Q,Li M Z,Leibham D,et al.The univector plasmid-fusion system,a method for rapid construction of recombinant DNAwithout restriction enzymes.Current Biology:CB,1998,8(24):1300-1309
[6]Reece-Hoyes J S,Walhout A J M.Gateway recombinational cloning.Cold Spring Harb Protoc,2018(1):1-6
[7]Park J,Throop A L,Labaer J.Site-specific recombinational cloning using gateway and in-fusion cloning schemes.Curr Protoc Mol Biol,2015,110(1):3.20.1-3.20.23
[8]韩占品,任健,于玮玮,等.花椰菜ERF114基因的克隆及Gateway技术构建表达载体.西南农业学报,2018,31(6):1128-1134Han Z P,Ren J,Yu W W,et al.Southwest China Journal of Agricultural Sciences,2018,31(6):1128-1134
[9]Li M Z,Elledge S J.MAGIC,an in vivo genetic method for the rapid construction of recombinant DNA molecules.Nat Genet,2005,37(3):311-319
[10]Garcíanafría J,Watson J F,Greger I H.IVA cloning:a single-tube universal cloning system exploiting bacterial in vivo assembly.Scientific Reports,2016,6(1):27459
[11]Wang Y,Liu Y,Chen J,et al.Restriction-ligation-free(RLF)cloning:a high-throughput cloning method by in vivo homologous recombination of PCR products.Genet Mol Res,2015,14(4):12306-12315
[12]Liu C J,Jiang H,Wu L,et al.OEPR cloning:an efficient and seamless cloning strategy for large-and multi-fragments.Scientific Reports,2017,7(1):44648
[13]Zhang Y,Werling U,Edelmann W.SLiCE:a novel bacterial cell extract-based DNA cloning method.Nucleic Acids Res,2012,40(8):e55
[14]Beyer H M,Gonschorek P,Samodelov S L,et al.AQUA cloning:a versatile and simple enzyme-free cloning approach.Plos One,2015,10(9):e0137652
[15]Li M Z,Elledge S J.SLIC:a method for sequence-and ligationindependent cloning.Methods in Molecular Biology,2012,852:51-59
[16]Jeong J Y,Yim H S,Ryu J Y,et al.One-step sequence-and ligationindependent cloning as a rapid and versatile cloning method for functional genomics studies.Applied&Environmental Microbiology,2012,78(15):5440-5443
[17]Yang J,Zhang Z,Zhang X A,et al.A ligation-independent cloning method using nicking DNA endonuclease.Biotechniques,2010,49(5):817-821
[18]Bitinaite J,Rubino M,Varma K H,et al.USER friendly DNAengineering and cloning method by uracil excision.Nucleic Acids Res,2007,35(6):1992-2002
[19]Geu-Flores F,Nour-Eldin H H,Nielsen M T,et al.USER fusion:a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products.NucleicAcids Res,2007,35(7):e55
[20]Nour-Eldin H H,Hansen B G,Norholm M H,et al.Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments.NucleicAcids Res,2006,34(18):e122
[21]Gibson D G,Young L,Chuang R Y,et al.Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods,2009,6(5):343-345
[22]Liu W,Ping Y,Tao W,et al.Homologous recombination-based DNA cloning methods and compositions:USA,US201213362382.2013-08-06.
[23]Sleight S C,Bartley B A,Lieviant J A,et al.In-Fusion BioBrick assembly and re-engineering.Nucleic Acids Res,2010,38(8):2624-2636
[24]Berrow N S,Alderton D,Owens R J.The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning.Methods Mol Biol,2009,498:75-90
[25]Zhu B,Cai G,Hall E O,et al.In-fusion assembly:seamless engineering of multidomain fusion proteins,modular vectors,and mutations.Biotechniques,2007,43(3):354-359
[26]Zeng F,Zang J,Zhang S,et al.AFEAP cloning:a precise and efficient method for large DNA sequence assembly.BMCBiotechnology,2017,17(1):81
[27]Liang J,Liu Z,Low X Z,et al.Twin-primer non-enzymatic DNAassembly:an efficient and accurate multi-part DNA assembly method.NucleicAcids Research,2017,45(11):e94
[28]Chen G J,Qiu N,Karrer C,et al.Restriction site-free insertion of PCR products directionally into vectors.Biotechniques,2000,28(3):498-500
[29]Van Den Ent F,Lowe J.RF cloning:a restriction-free method for inserting target genes into plasmids.Journal of Biochemical and Biophysical Methods,2006,67(1):67-74
[30]Zeng F,Hao Z,Li P,et al.A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends.BMC Biotechnology,2017,17(1):32
[31]Quan J,Tian J.Circular polymerase extension cloning of complex gene libraries and pathways.Plos One,2009,4(7):e6441
[32]Quan J,Tian J.Circular polymerase extension cloning for highthroughput cloning of complex and combinatorial DNA libraries.Nature Protocols,2011,6(2):242-251
[33]Bryksin A V,Matsumura I.Overlap extension PCR cloning:a simple and reliable way to create recombinant plasmids.Biotechniques,2010,48(6):463-465
[34]Jajesniak P,Wong T S.QuickStep-Cloning:a sequenceindependent,ligation-free method for rapid construction of recombinant plasmids.Journal of Biological Engineering,2015,9:15
[35]Qaidi S E,Hardwidge P R.ABC cloning:an efficient,simple,and rapid restriction/ligase-free method.MethodsX,2019,6:316-321
[36]Peleg Y,Unger T.Application of the Restriction-Free(RF)cloning for multicomponents assembly.Methods Mol Biol,2014,1116:73-87
[37]Unger T,Jacobovitch Y,Dantes A,et al.Applications of the restriction free(RF)cloning procedure for molecular manipulations and protein expression.Journal of Structural Biology,2010,172(1):34-44
[38]Bandyopadhyay B,Peleg Y.Facilitating circular permutation using restriction free(RF)cloning.Protein Engineering,Design&Selection:PEDS,2018,31(3):65-68
[39]Dai C,Miao C X,Lu G X.Site-directed mutagenesis based on overlap extension PCR.Progress in Modern Biomedicine,2010,10(03):177-192
[40]Guo Z Y,Hao X H,Yan J L,et al.A new method of site-directed mutagenesis by modifying overlap extension PCR.Biotechnology,2009,19(6):34-36
[41]Bond S R,Naus C C.RF-Cloning.org:an online tool for the design of restriction-free cloning projects.Nucleic Acids Research,2012,40(W1):W209-W13
[42]Steffens D L,Williams J G.Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.J Biomol Tech,2007,18(3):147-149
[43]Yang Y,Huang L,Wang J,et al.Expression,characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus.Enzyme and Microbial Technology,2015,68:43-49
[44]Tseng W C,Lin J W,Wei T Y,et al.A novel megaprimed and ligasefree,PCR-based,site-directed mutagenesis method.Analytical Biochemistry,2008,375(2):376-378
[45]Taniguchi N,Murakami H.Multiple site-directed and saturation mutagenesis by the patch cloning method.Methods in Molecular Biology,2017,1498:339-347
[46]Taniguchi N,Nakayama S,Kawakami T,et al.Patch cloning method for multiple site-directed and saturation mutagenesis.BMC Biotechnology,2013,13(1):1-8
[47]Zeng F,Zhang S,Hao Z,et al.Efficient strategy for introducing large and multiple changes in plasmid DNA.Sci Rep,2018,8(1):1714
[48]Erijman A,Dantes A,Bernheim R,et al.Transfer-PCR(TPCR):a highway for DNA cloning and protein engineering.Journal of Structural Biology,2011,175(2):171-177
[49]Steinmetz E J,Auldridge M E.Screening fusion tags for improved recombinant protein expression in E.coli with the Expresso?;solubility and expression screening system.Current Protocols in Protein Science,2017,5(27):1-20
[50]Berrow N S,Büssow K,Coutard B,et al.Recombinant protein expression and solubility screening in Escherichia coli:a comparative study.Acta Crystallographica Section D,Biological Crystallography,2010,62(10):1218-1226
[51]Perrakis A,Romier C.Assembly of protein complexes by coexpression in prokaryotic and eukaryotic hosts:an overview.Methods Mol Biol,2008,426:247-256
[52]Romier C,Ben Jelloul M,Albeck S,et al.Co-expression of protein complexes in prokaryotic and eukaryotic hosts:experimental procedures,database tracking and case studies.Acta crystallographica Section D,Biological Crystallography,2006,62(10):1232-1242
[53]陆路,熊文,张钰琨,等.RF克隆结合巢式PCR构建金黄色葡萄球菌组氨酸蛋白激酶AgrC表达载体.中国生物工程杂志,2014,34(10):55-60Lu L,Xiong W,Zhang Y K,et al.Journal of Chinese Biotechnology,2014,34(10):55-60
[54]Li M Z,Elledge S J.Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.Nat Methods,2007,4(3):251-256
[55]Hartley J L.Use of the gateway system for protein expression in multiple hosts.Current Protocols in Protein Science,2003,5(17):1-10
[56]Busso D,Delagoutte-Busso B,Moras D.Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli.Analytical Biochemistry,2005,343(2):313-321
[57]Moreland N,Ashton R,Baker H M,et al.A flexible and economical medium-throughput strategy for protein production and crystallization.Acta Crystallographica Section D,Biological Crystallography,2005,61(10):1378-1385