Seamless cloning and gene fusion
Quinn Lu
Gene Expression and Protein Biochemistry,Discovery Rearch,GlaxoSmithKline,Mail Code:UE0548,709Swedeland Road,King of Prussia,PA 19406,USA
Gene fusion technology is a key tool in facilitating gene function studies.Hybrid molecules in which all the com-ponents are joined precily,without the prence of intervening and unwanted extraneous quences,enable accurate studies of molecules and the character-ization of individual components.This article reviews situations in which amlessly fud genes and proteins are required or desired and describes molecular approaches that are available for generating the hybrid molecules.
Introduction
With the completion of various genome quencing pro-jects,functional analysis of gene products has taken the stage.Gene fusion technology has an important role in many aspects of gene function studies,including gene and protein tagging,reporter gene studies,domain swapping studies,mutagenesi
s studies and gene knock-in and/or knock-out experiments [1].Classic gene fusion techniques involving type II restriction enzyme digestion and DNA ligation reactions (the so-called cut-and-paste reactions)have been ud as standard procedures for generating hybrid genes.However,such procedures often leave behind operational quences,such as restriction sites,at the junction.The unwanted quences can change the spacing between the DNA elements and introduce extra amino acid residues at the junction,which could have undesired effects on the structure and activity of the fusion protein and could therefore interfere with accurate study of the fusion gene.This review discuss examples in which the u of precily fud genes is required or desired and outlines approaches that can be ud to achieve amless gene fusion.Owing to the lack of reviews in this area some historical information together with recent advances will be discusd here.
Seamless gene fusion and applications
Seamless cloning and gene fusion are process that allow two or more DNA fragments to be joined precily so that no unwanted nucleotides are added at the junctions between DNA fragments.This is the ideal situation for generating hybrid genes and the following ction highlights some examples that show the importance of amless gene fusion.Promoter and exon studies
Gene promoters contain arrays of regulatory elements to which transcriptional factors bind and interact with each
other to regulate transcription.Promoter deletion analy-sis allows identification of the functional elements and provides crucial information on the mechanism of gene regulation.However,becau spacing between various regulatory elements is often important,a linker of the same length is usually required to prerve the spacing and helical facing of the elements.Linker scanning ana-lysis [2](Box 1,Ca 1)of gene promoters requires am-less DNA fusion or quence replacement.Molecular evolution approaches,such as exon and DNA shuffling,for producing proteins with desired biochemical and/or biophysical properties also require amless splicing of various functional elements [3].In eukaryotic cells,chimeric genes and/or proteins can be created by intron-mediated RNA splicing [4,5].In the experiments synthesis of RNA substrates and/or exon-tagged ribozymes requires careful design and generation of the chimeric precursor genes.As long as the hybrid gene is generated correctly ,amless fusion can be achieved on splicing.
Protein functional studies and protein engineering
Proteins are compod of functional domains.To elucidate the function of a particular domain in a de
fined protein,mutant proteins with the domain deleted or replaced by a similar domain from a homologous protein are often needed.Such a domain deletion or swapping experiment would benefit greatly from amless fusions of the rele-vant parts to eliminate potential negative effects caud by the prence of operational quences.In more general terms,amless fusions of various domains are esntial for protein engineering,including the generation of novel hybrid molecules and antibody engineering.In the ca of antibody engineering,grafting of the complementarity-determining region (CDR)from mou antibodies to human frameworks (CDR-grafting)and site-directed mutagenesis (SDM)are routine procedures [6].Chimeric proteins can also be generated via intein-mediated protein splicing and ligation [7].As long as the intein-containing precursor proteins are generated amlessly,preci protein fusions can be achieved.
Protein production
Protein production has been improved by the u of tags and fusion partners.They confer solubility and stability and facilitate subquent affinity purification of the target protein [1].In cas in which the activity of a particular protein is unaffected by the prence of its fusion partner or a tag,the entire fusion protein is ud in subquent applications.This is often the ca for enzymatic studies and assay development.However,in many cas
removal
Corresponding author:Lu,Q.(quinn.2.).
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0167-7799/$-e front matter Q 2005Elvier Ltd.All rights rerved.doi:10.1016/j.tibtech.2005.02.008
of the fusion partner is required or desired and can be achieved via an engineered protea cleavage site.This process requires a amless junction between the protea cleavage site and the protein of interest.For structural studies,in some cas fusion proteins with a short tag (such as a hexa-Histidine tag)have been successfully crystallized and removal of the tag is unnecessary.How-ever,for tho fusion proteins with a tag that failed to crystallize,the impact of the tag is hard to asss and its removal is usually desirable.
In producing mature and active proteins,proteins with native amino acid residues are often required.This is exemplified by proteins such as RANTES (Regulated on
Activation,Normal T Expresd and Secreted),inter-leukin-18(IL-18),IL-1b and hirudin [8–11].Addition of a methionine residue at the N-termini of the proteins significantly reduces activity.In the ca of RANTES,Met-RANTES was found to behave as an antagonist for the authentic RANTES [8].A practical approach for producing proteins with authentic N-terminal residues li is to express and purify them as fusion proteins with an N-terminal tag.Following purification,and refold-ing if nece
ssary,the tag and any unwanted amino acid residues are then removed via a specifically engineered protea cleavage site.This process requires a amless junction between the protea cleavage site and the pro-tein of interest.Enterokina [12],factor Xa [13]and ubiquitin-specific proteas (Ubps)[14,15]that cleave at the C-terminal of their recognition quences are often ud for this purpo.The tobacco etch virus (TEV)pro-tea [16]can also be ud becau it has a relaxed requirement on the amino acid residue immediately following the cleavage site (the P10position).The ubiquitin/Ubp system is probably the first tagging system that is ud to generate proteins with native N-terminal residues both in vivo and in vitro (Box 1,Ca 2).In producing mature human Apo A-I li ,Moguilevsky and colleagues [17]found that the ubiquitin tagging system is one of the most straightforward methods.
Genome manipulation我爱家乡我爱祖国
Targeted gene knockout and knock-in technologies in model organisms provide powerful genetic tools for gene function analysis in vivo .This process often involves deletion of the entire open reading frame (ORF)or a part that encodes a particular domain,and in some cas replacement of the with quences of a mutant allele or an orthologue.The studies require generation of knockout and knock-in con-structs in which all the functional elements,including flanking regions,req
uired for homologous recombination are precily joined.Seamless gene fusion technologies would greatly facilitate construct generation for such studies.Link and colleagues [18]ud the overlap PCR technology (e following ction)to generate constructs with preci gene deletions for functional studies in li genome.
For molecular and pathogenesis studies of animal virus and vaccine vector development,it is often necessary to synthesize the entire viral genome or to engineer hybrid viral vectors.Using a amless gene fusion technique,Yount and colleagues [19]have successfully asmbled a 31.5Kb recombinant viral genome from multiple PCR fragments (Box 1,Ca 3).
Methods for achieving amless gene fusion
Before PCR was invented,amless fusion of DNA frag-ments was achieved by complicated procedures routinely involving the u of bacterial phage M13-bad site-directed mutagenesis,oligonucleotide primers and linkers and exonucleas,followed by plasmid propagation li .In some cas it was achieved using RecA-dependent homologous recombination.The process required careful design of the target constructs and involved a laborious procedure.Given the difficulties
Box 1.Seamless gene fusion ca studies
Ca 1.Scanning mutagenesis.Scanning mutagenesis experiments were designed to identify cis regulatory elements in gene promoters [2]or to study crucial amino acid residue(s)in protein structure and function.For a gene promoter,a linker scanning analysis is performed.A panel of DNA constructs is made and within each a gment of the promoter is replaced by a defined linker fragment of the same length to restore the spacing.For a protein,alanine scanning mutagenesis is often performed [65]in which a t of mutants are generated with each charged amino acid residue mutated to alanine.Traditionally,scanning mutagenesis constructs were generated by a laborious process involving the u of exonucleas,linkers and liga [2]or by bacterial phage M13-bad site-directed mutagenesis [20].With the advent of PCR,the variant constructs can be easily generated by overlap PCR [66]or in vivo recombination [57,58].
Ca 2.The ubiquitin tagging system and applications.Ubiquitin is a highly conrved eukaryotic protein of 76amino acid residues that is naturally expresd as polyubiquitin,which is then cleaved precily in vivo at the C-terminus of the ubiquitin moiety by a family of ubiquitin processing proteas (Ubps).The amino acid residue at P10could be any residue except proline.The high specificity and the unique cleavage fashion of Ubps have made the ubiquitin–Ubp pair an ideal taggi
ng system for gene expression in eukaryotic and prokaryotic cells [67].To study the impact of the N-terminal amino acid residue on stability of proteins in yeast,Varshavsky and colleagues [21,22]created a t of ubiquitin–X–b gal amless fusion genes,in which X is a codon for one of the 20amino acids.Upon expression in yeast,the nascent fusion proteins are de-ubiquitinated by Ubps in vivo ,exposing the X–b gal proteins with various N-terminal residues.Stability studies of the X–b gal proteins in yeast led to the discovery of the N-end rule of protein stability in vivo [68].Co-translational processing has also allowed protein production in eukaryotic cells [67,69].In prokaryotic cells,ubiquitin fusion proteins are not procesd in vivo owing to the lack of Ubps.After purification of the fusion protein,the ubiquitin tag can be removed with a purified Ubp in vitro [15].Along the same line,the yeast small ubiquitin-like modifier (SUMO)protein,SMT3,has been ud as a fusion tag for protein production in E.coli [14](/r_and_d/protein_expression.php3).This tag can be removed by treating with SUMO hydrola (ubiquitin-like protea 1)in vitro .The ubiquitin–Ubp proteins thus provide a unique system for producing proteins with an authentic N-terminus,as long as the ubiquitin-ORF fusion is generated amlessly.
Ca 3.Viral genome asmbly.In an effort to asmble a 31.5kb full length infectious cDNA of a recombinant coronavirus,Yount and colleagues ud a amless gene fusion approach involving PC
R and the u of a type IIS restriction enzyme [19].To do so,the viral genome was PCR amplified as ven neighboring fragments using primers with an Esp 3I cleavage site engineered at the ends.Esp 3I digestion removes any unwanted nucleotide residues at the ends of the PCR fragments,leaving specific four ba overhangs that are complementary for the neighboring gments.A stepwi ligation reaction allowed directional asmbly of the full-length viral genome.
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with the classical approaches,only a few examples of amless gene fusion using the methods can be found. One such example is the generation of a t of mutant constructs ud for link-scanning analysis of gene pro-moters(Box1,Ca1)[2,20]and another for protein stability studies in yeast[21,22](Box1,Ca2).
With the advent of PCR technology,amless gene fusion has been enabled.Several methods for this purpo have been created,and many other approaches could be modified to achieve the purpo
.The methods are dis-cusd below and summarized in Table1.PCR is always involved,either to allow preci DNA manipulation or to modify the ends of the DNA elements for appropriate gene fusion.With the innovative approaches,our ability to manipulate DNA quences has been taken to an unpre-cedented level.For example,in a study on the influence of P10amino acid residue on enterokina(EK)cleavage in a fusion protein,a t of20variant GST–EK–X–calmodulin fusion genes(in which X is one of the20amino acid residues and GST is glutathione S-transfera)were created by a amless gene fusion technique via PCR and ligation-independent cloning[12].
Overlap PCR
Overlap PCR(Figure1)was described shortly after the invention of PCR[23,24].It is a robust process that is independent of any restriction sites;any two fragments can be freely joined at any predetermined quence location,provided that the fragments can be faithfully amplified.With the same principle,multiple DNA frag-ments can be spliced together amlessly[25]and recombinant fusion genes as long as20Kb have been obtained[26].Overlap PCR has been ud to introduce point mutations,inrtions,deletions and replacements into any point of a gene in a amless fashion[23,24].The PCR generated fusion genes can subquently be cloned
Table1.Methods for achieving amless gene fusion
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Methods Applications Pros Cons
Gene synthesis Creation of hybrid or novel genes at
will.Generation of DNA fragments
with any desired changes.Allow fusion gene designing with
accuracy at ba pair(bp)level.
Allow codon optimization of any
open reading frame.
Practical when the hybrid gene is!500bp
in length,although longer is possible.
Overlap PCR Asmbly of multiple DNA
fragments.Directional DNA asmbly,versatile
and efficient,independent of
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restriction sites.
Requires multiple PCR reactions.The
longer the hybrid product is,the higher risk
for PCR introduced errors.Reasonable for
hybrids!10Kb,although longer is
英文信件开头possible.
Inver PCR Prepare vector backbones with
desired terminal quences for
applications listed below.Can also
be ud to introduce point mutation,
deletion,inrtion and quence
replacement on a circular plasmid.Ideal for preparing vectors with
desired terminal quences for
amless cloning.
The vector backbone might contain
quence errors introduced by PCR.The
longer the vector backbone,the higher risk
for PCR introduced errors.
QuickChange e site-directed mutagenesis Point mutation,deletion,inrtion
and quence replacement on any
plasmid backbones
Most widely ud method with a
ries of kits commercially available.
Multiple mutations in a single
reaction possible.总有一天出头日
Vector size O8Kb may have a decread
efficiency,however,template up to19Kb
has been ud().
Type IIS restriction enzyme-mediated gene fusion Asmbly of long multiple DNA
fragments.
Directional ligation of multiple PCR
fragments to asmble O15Kb
fusion genes or viral genomes.
Requires multiple PCR reactions and
restriction digestion.For fusion of a PCR
fragment with an existing fragment already
contained in a vector,special vector is
required.
Ligation-independent cloning Fusion of a PCR fragment with a DNA
element(s)contained in a vector.
Directional cloning,independent of
restriction sites and the u of liga.
Need a specially designed LIC vector.The
inrt and vector DNA fragments need to be
treated to generate single-stranded
overhangs.
In-Fusion e cloning Fusion of a PCR fragment with a DNA
element(s)contained in a vector.Directional cloning,independent of
restriction sites and the u of liga.
Mechanism of action not disclod by the
supplier.The In-Fusion e enzyme is
relatively expensive.Seamless only when
the vector fragment doesn’t contain extra
quence at the fusion junction.
RecA-dependent recombination Allelic replacement li.
Construction of recombinant
adenoviral genomes li.
Intermolecular recombination
百科知识大全between two circular molecules
possible.
Work in recA C strains and requires long
(O1Kb)homologous arms.A low
frequency event,usually requires lection
and/or conterlection.
RecA-independent recombination Point mutation,deletion,inrtion in
Point mutation,deletion,inrtion.
Work in commonly li
strains such as DH5a and JM109
(recA strains),only requires O12bp
homologous arms.
Mechanism of action unclear,although
independent of RecA.Most efficient if the
origin of replication and a lectable marker
is contained in a parate fragment.
Red/ET recombination Point mutation,deletion,inrtion.
Subcloning from a complex source
Allelic replacement li.
Requires30–50homologous arms.
Vector fragments generated by
inver PCR have been ud.
Work in recBC sbcA mutant strains such as
JC8679or in recBC C strains overexpressing
RedE/RedT/Red g
Gap repair in yeast Gene fusion and subcloning in yeast.Requires O30bp homologous arms,
vector fragments can be generated
by inver PCR.Requires a yeast origin of replication and a lectable marker in the backbone.
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into appropriate vectors for downstream applications.Note that a special ca of overlap PCR is gene synthesis,in which overlapping oligonucleotides are ud as PCR templates for gene asmbly [27].This approach can be readily ud if there is no suitable template DNA that can be ud for PCR amplification,and/or if the hybrid gene is relatively short (!500bp).
Site-specific mutagenesis
Site specific mutagenesis,including point mutations,inrtions,deletions and replacements,is carried out on a circular plasmid template that contains the target gene.A variety of mutagenesis approaches are available for generating point mutations [28].However,mutagenesis tasks involving deletion,inrtion or replacement of a quence are typically achieved by a PCR-bad method.Inver PCR us two oppositely positioned primers to amplify a plasmid backbone,which allows generation of point mutations,deletions and inrtions at any location of the plasmid [29].However,the PCR step could poten-tially introduce quence errors in the circular plasmid backbone.This issue was addresd by the QuickChange e ( )mutagenesis technology [30,31].As illustrated in Figure 2,the method relies on the u of two fully complementary
mutagenic primers and Pfu DNA polymera.Given that the primers are completely complementary to each other,and that Pfu lacks strand-displacement activity,the mutant strands are only linearly amplified from the original template DNA during
the multiple thermal cycles.This design thus prevents amplification of any errors that occurred during DNA synthesis.The QuickChange e procedure and improve-ments have been ud to generate a DNA inrtion,deletion or quence replacement at any site of the template DNA [32,33]and even to introduce multiple mutations into a template simultaneously [34].The QuickChange e mutagenesis kit thus allows easy manipu-lation of any circular plasmid DNA with high fidelity.The u of type IIS restriction enzymes
Type IIS restriction enzymes are a class of enzymes that cleave outside of their recognition quences,for example Sap I,Bsa I and Fok I [35].They have been ud to generate cohesive ends from PCR fragments for amless asmbly of genes and viral genomes [19,36,37](Figure 3a).In cas in which one of the two fragments is a generic one to which multiple partners will be fud,such as a purification tag (e.g.malto binding protein)for protein production,the fragment is usually inrted into the expression vector first.The vector is then made to accept its fusion partner through amless cloning [38,39](Figure 3b).EK vector of Stratagene (www.stratagene.c
om )is li expression vector that contains the T7/lacO–CBP–EK–MCS expression castte (CBP is calmodulin binding peptide and MCS is multiple cloning site).Cloning via the type IIS enzyme Eam 1104I allows amless fusion of the target ORF immediately downstream of the CBP–EK ORF.After expression and purification of the CBP–EK–ORF fusion protein,removal of the CBP–EK polypeptide with enterokina allows recovery of the target recombinant protein with native amino acid quence [39].The IMPACT e vectors pTYB and pTWIN ( )allow amless fusion of ORFs for a target protein with an intein via Sap I for protein production and subquent protein splicing.
Type IIS restriction sites have also been ud in pre-paring acceptor vectors for amless cloning [38,40]allow-ing a DNA fragment to be fud amlessly with existing DNA fragments contained in the vector.This is achieved by inver PCR of the plasmid backbone with a type IIS restriction site engineered in the primers.Subquent restriction digestion generates the vector with desired compatible cohesive ends.It should be noted that the type IIS restriction enzyme-bad approaches involve restric-tion enzyme digestion of both the vector and the inrted DNA fragments.In cas in which the DNA fragments contain internal sites for a particular type IIS enzyme in u,the methylation inhibition approach can be ud to block digestion of the internal sites by the enzyme [3
8,41]or a different type IIS enzyme is ud to produce DNA fragments with desired cohesive ends,if possible.The vector and inrt fragments can be asmbled as illu-strated (Figure 3a).Alternatively,the desired circular plasmid can be asmbled from multiple PCR fragments using the strategy outlined in Figure 3a.
Ligation-independent cloning (LIC)
To circumvent limitations of the classical cut-and-paste methods for generating hybrid genes,LIC was developed to clone PCR fragments independent of restriction enzyme
Figure 1.Seamless gene fusion by overlap PCR.The diagram shows amless fusion of DNA fragments X and Y.The two DNA fragments are PCR amplified individually.Primers P2and P3are designed so that the 50-end 15bas are complementary to each other.The PCR products are then ud as templates for a cond PCR amplification with primers P1and P4.The complementary part of P2and P3could be part of fragment X or fragment Y.Note that to facilitate efficient PCR amplification,the melting temperature (T m )for all primers should be made to be similar within the range of 558C–758C.
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digestion and ligation[42].The method relies on the u of DNA polymeras with30to50exonuclea activities (T4or Pfu polymeras)to create12ba50overhangs on DNA fragments for annealing(Figure4).With its unique properties,LIC has been ud to achieve amless cloning to join ORFs encoding protein domains for protein pro-duction[12,39,43,44].Using chimeric PCR primers con-taining ribonucleotides,LIC ends with cohesive overhangs can also be generated by treating the
PCR products with other reagents,such as uracil DNA glycosyla[45]or rare-earth metal ions[46].Jarrell and colleagues[47]ud primers with three concutive ribonucleotide bas or a single20-O-methyl ribonucleotide to terminate DNA syn-thesis at the complementary strand,thus generating single-stranded overhangs during PCR.Given the simpli-city,this method can be ud to facilitate high-throughput cloning experiments.
The In-Fusion e PCR cloning system()allows cloning of a PCR fragment into any linearized vector,as long as the PCR fragment contains 15bp arms homologous to tho in the vector fragment [48].However,the exact components of the enzyme(s) involved in this reaction are not disclod by the company. Becau the terminal ends are crucial for this reaction,the vector fragment needs to be specially prepared to achieve amless fusion of the inrt with an existing ORF in the vector.The vector fragment can be prepared using inver PCR,or by treating with a type IIS restriction enzyme to remove unwanted nucleotides[40].The In-Fusion e sys-tem is so far the most straightforward system for PCR cloning,it should enable automation of PCR cloning.
In vivo recombination
Homologous recombination allows exchange of genetic material between two molecules with homol
ogous quence regions.Becau the location for homologous recombination can be freely chon,it allows amless DNA manipulation.Traditionally,this process is ud in allelic replacement for gene function studies li and yeast cells.This is usually achieved by a two-step homo-logous recombination process[18,49].A circular plasmid bearing a positive lectable marker and a negative lect-able marker isfirst incorporated into a specific site of the target ORF on the chromosome or an episomal vector. Recombinants are lected through the positive lection marker.The lection fragment is subquently replaced with another fragment containing a modified version of the ORF by counter lection against the negative lect-able marker.This process allows preci and amless gene manipulation on chromosome(s).In yeast,manipulation of
Figure2.Seamless DNA manipulation by QuickChange e site-directed mutagenesis.The diagram shows steps involved in site-directed mutagenesis for generating point mutations(a),inrtions(b)or deletions(c).In all the cas,two complementary mutagenic primers(or megaprimers in ca b)are ud with each having O15ba homologous quencesflanking the mutagenic site.After primer extension cycles with Pfu polymera,the undesired methylated template DNA and mi-methylated hybrids are fragmented by treating with restriction enzyme Dpn I.The desired mutant circular duplexes are recovered li following transformation.The plasmid backbone contains an origin of replication(ori)and a lectable marker(sm).
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genes carried on a plasmid is traditionally achieved by a one-step gap repair process,which requires only w 30bp homologous arms [50].Using PCR technology,the hybrid constructs for such gap repair experiments can easily be generated to achieve deletion,inrtion and quence replacement.A yeast–E.coli –mammalian shuttle vector was ud for generating hybrid fusion genes amlessly in Saccharomyces cerevesiae followed by plasmid rescue li [51].
Homologous recombination li can be achieved by three different mechanisms:RecA-depende
nt,RecA-independent and Red/ET-dependent.RecA binds to single-stranded gments of DNA and promotes strand invasion and exchange between homologous quences.RecA-mediated recombination requires a long homologous region (O 1Kb)and occurs at a low frequency [52].It has been ud to generate recombinant adenoviral genomes [53]and in the two-step allelic replacement experiment to modify genes carried on bacterial artificial chromosomes (BACs)and P1-derived artificial chromosomes (PACs)[54,55].The RecA-independent pathway works in recA strains and requires the two DNA fragments to be recom-bined to have O 12bp homologies at the ends [56–58](Figure 5).Co-transformation of the two linear DNA fragments allows recovery of circular plasmids.However,the exact mechanism for this reaction is unclear and the cloning efficiency can be low.A high cloning efficiency is obtained when the origin of replication and a lectable marker are contained in a parate fragment to enforce recombination [57,58].This strategy has been ud to
generate a ries of cysteine mutations of a protein in a high-throughput fashion [59].The Red/ET system only requires 30–50bp homologous regions between a linear vector fragment and a linear inrt fragment for efficient recombination li (Figure 5).The technology relies on the function of the Red a /Red b protein pair that of l prophage,or their functionally equivalent RecE/RecT protein pair of Rac prophage,in which Red a and RecE are 50-30exonucleas and Red b and RecT are sin
gle-stranded DNA annealing proteins [52,61].In recBC sbcA mutant strains,the RecBCD exonuclea activity is inactivated and the RedE/RedT proteins are expresd from a cryptic Rac prophage that is activated by the sbcA mutation,homologous recombination between linear fragments is possible [52,60,61].In recBC C strains,homologous recom-bination between linear fragments can be enabled by over-expressing RedE/RedT proteins and the Red g gene product,which inhibits RecBCD exonuclea activity [51,60]”.The RecA-independent system and the Red/ET system are thus functionally comparable to the gap repair system in yeast cells.DNA fragments with the short arms can be generated by PCR and ud directly in the in vivo recom-bination.li gap repair approaches have been ud in construction generation for gene expression,clon-ing genes from complex sources and in achieving allelic replacement on chromosomes [52,61].
Concluding remarks
Seamless cloning and gene fusion is the ideal situation for creating hybrid DNA molecules for all applications.
Figure 3.Seamless gene cloning and gene fusion via a type IIS restriction enzyme.Sap I is ud as an example.(a)Seamless asmbly of fragments X,Y,and Z.The fragments are first individually PCR amplified,with primers containing a Sap I site.The primers are so designed that upon Sap I digestion specific cohesive ends are generated for each fragment.Following Sap I digestion,ligation of the fragments results in amless asmbly of X,Y,and Z.The 50-end of X and the 30-end of Z are made
to contain Sap I (with ends incompatible to other ends of X,Y and Z)or any other restriction sites for further subcloning.(b)Seamless fusion of fragment X with fragments Y and Z contained in a vector.Fragment A is PCR amplified with primers containing a Sap I site.The primers are so designed that upon digestion with Sap I cohesive ends are generated.The vector fragment,which contains fragments Y and Z,was specially engineered and prepared by Sap I digestion.Ligation of the Sap I-treated fragment A with the vector fragment results in amless fusion of X with Y and Z.The plasmid backbone contains an origin of replication (ori)and a lectable marker (sm).Note that the nucleotide bas comprising the cohesive ends can be part of the fragment X or its fusion partners,and can be chon to make ligation of the fragments directional.
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