BACKGROUND
the manipulation of recombinant dna molecules is an indispensable step in modern high-throughput protein crystallization studies  <cit> .
restriction enzyme/ligase cloning, which relies on restriction enzyme digestion and ligation, is a simple and easy way to move a fragment of double-stranded dna from one plasmid to another  <cit> .
however, this technique has two limitations  <cit> : it is ineffective when lack of unique restriction sites and sometimes results in introduction of unwanted extra sequences.
to circumvent these limitations, various restriction endonuclease cleavage site–independent cloning methods have recently been developed .
these methods have made cloning more accessible in cases in which conventional restriction site cloning was difficult or impossible.
while as alternative cloning strategies are still required for more choices.
the restriction-free  cloning strategy, as described extensively in the literature, was developed as a powerful tool for reconstituting genes in circular vectors  <cit> .
because rf cloning requires no alterations in the plasmid or the gene of interest, it is exceptionally well-suited for high-throughput cloning.
the gene of interest is amplified in a regular polymerase chain reaction , which produces a primer pair that, once annealed to the vector of interest, is extended in a linear amplification reaction.
thus, this method relies on amplified genes functioning as primers.
however, this approach also has limitations.
first, the motion of large dna fragments and the formation of secondary structures will affect the efficiency of pcr.
second, this method relies on digestion with dpni, which cleaves methylated dna, to remove parental plasmids.
the efficiency of dpni treatment is influenced by many factors, and requires vector propagation in dam+ strains  <cit> .
in this paper, we describe a simple and fast method for performing gene reconstitution by modified restriction-free  cloning.
in this method, two rounds of pcr generate two dna fragments with compatible 5' or 3’ cohesive ends, which are therefore able to ligate to each other.
this new method is independent of the existence of restrictions sites and dpni treatment.
using this method, we made  <dig> constructs with inserts of variable size, with average cloning efficiency higher than 85%.
the efficiency was not significantly affected by the insert length up to 20 kb.
RESULTS
method overview and primer design
figure  <dig> shows the scheme for mrf cloning.
we define the 5–8 bp dna fragments before the insert site a as “5’ overhang” and those after insert site b as “3’ overhang” .
to replace a gene  in a vector between sites a and b, we designed eight primers : primer  <dig>  forward primer, which contains a ~25 bp sequence homologous to the positive strand of the gene; primer  <dig>  reverse primer, which contains a ~25 bp sequence homologous to the negative strand of the gene; primer  <dig>  forward primer, which is the same as primer  <dig> but has an additional 5’ overhang at the 5’ end; primer  <dig>  reverse primer, which is the same as primer  <dig> but has an additional 3’ overhang at the 5’ end; primer  <dig>  forward primer, which contains a ~25 bp sequence homologous to the negative strand of the vector; primer  <dig>  reverse primer, which contains a ~25 bp sequence homologous to the positive strand of the vector; primer  <dig>  reverse primer, which is the same as primer  <dig> but has an additional 5' overhang at the 5’ end; primer  <dig>  reverse primer, which is the same as primer  <dig> but has an additional 3’ overhang at the 5’ end.
all primers were designed with g or c as the 5’ and 3’ terminal nucleotide.fig.
<dig> schematic representation of mrf cloning.
the insert gene or vector was amplified by regular double-primer pcr using genomic dna, cdna, or the original vector as template.
compatible cohesive ends of insert gene or vector were created by two single-primer linear pcrs performed in parallel, followed by annealing of the two pcr products.
inserts and acceptors with compatible cohesive ends were then assembled by ligation
we used two pairs of primers, primer 1/primer  <dig> and primer 5/primer  <dig>  to generate two dna fragments, using the target gene and vector as templates, respectively.
the resultant pcr products were gel purified.
we then amplified these two dna fragments in two separate pcr reactions containing either forward or reverse primer, which will add 5’ overhang or 3’ overhang to the pcr products .
finally, dna fragments with complementary overhang at the 5’ or 3’ end were able to anneal to each other, and were joined by dna ligase .
the ligated products were then transformed into dh5α competent cells.
the inserted genes were verified by colony pcr and further confirmed by dna sequencing.
mrf cloning can assemble insert dna fragment into target vector
we first tested this protocol to reconstitute the e. coli rada gene into pet22b.
based on the initial success of the protocol, we continued to employ it to generate the constructs needed for our studies.
for example, we planned to replace the rada gene in vector pet22b between the start codon atg  and the sequence caccaccaccaccaccac   to yield a new construct with the gene under the control of a t <dig> promoter and a c-terminal his6-tag to facilitate protein purification.
as shown in fig.
<dig>  to replace the rada gene in the vector, two parallel pcrs were performed to amplify each dna fragment using the primer pairs pet22b1/pet22b <dig> and rada1/rada <dig>  as shown in additional file 3: table s <dig>  using pet22b or e. coli genomic dna as templates to generate dna fragments “1” and “2”.
amplified products were separated by 1% agarose gel electrophoresis and purified by gel extraction.
we then performed two separate single-primer linear pcrs:  using pet22b <dig> or pet22b <dig> alone, with dna fragment “1” as the template, to obtain single-strand dna fragment “3-1” or “3-2”; and  using rada <dig> or rada <dig> alone, with dna fragment “2” as template, to obtain single-strand dna fragment “4-1” or “4-2”.
fragments “3-1” and “3-2” and “4-1” and “4-2” were then annealed to obtain double-strand dna fragments “3” and “4”, which have sticky ends that can ligate with other compatible ends.
for single-primer pcr, we used ~500 ng of template, about  <dig> times more than the standard amount recommended for double-primer pcr , as dna amplification in single-primer pcr is linear .
as the efficiency of cohesive ligation is higher than that of blunt-end ligation, the parental dna of the products of second-round pcr did not need to be removed.
after pcr purification, these second-round pcr products were ready for ligation.
in each transformation, we routinely checked eight colonies at random from each transformation by colony pcr with a forward primer annealing to vector and a reverse primer annealing to the inserted gene.table  <dig> pcr reaction components
phusion high fidelity dna
the agarose gel in fig.
<dig> shows the dna products of one sample at successive steps of our procedure.
plasmid alone, prior to pcr, shows two major bands .
after the double-primer pcr with forward  and reverse  primers, major bands can be seen at the expected sizes corresponding to the pcr-synthesized linear dna .
single-primer pcr with forward  or reverse  yielded bands at the expected size of ~ <dig>  kb  and ~ <dig>  kb  after annealing the products of single-primer pcr, representing the pcr-synthesized linear dna with cohesive ends.
additional smaller bands represent non-specific pcr products or single-strand dna.
the ligation of insert into plasmid vector is performed by t <dig> dna ligase using a molar ratio of 1: <dig> vector to insert.
as shown in fig.
<dig>  lane  <dig>  and  <dig> , insert and vector were ligated to one another and shifted to a higher molecular weight.
the inserted genes were amplified by colony pcr.
the presence of forward and reverse cloning sites were confirmed by dna sequencing .fig.
<dig> gel electrophoresis separation of double-primer and single-primer pcr products.
1: parental plasmid pet22b alone; 2: pcr product from reaction with primers pet22b1/pet22b <dig> using plasmid pet22b as template; 3: pcr product from reaction with primers rada1/rada <dig> using e. coli genomic dna as template; 4: pcr product from reaction with primers genecluster3-1/genecluster3- <dig> using e. coli genomic dna as template; 5: annealed pcr products from two single-primer linear reactions using primer pet22b <dig> or pet22b <dig>  and the dna sample from lane  <dig> as template; 6: annealed pcr products from two single-primer linear reactions using the primer rada2fw or rada2rv, and the dna sample from lane  <dig> as template; 7: annealed pcr products from two single-primer linear reactions using the primer genecluster3- <dig> or genecluster3- <dig>  and the dna sample from lane  <dig> as template; 8: mixture of dna samples from lanes  <dig> and  <dig> in a molar ratio of 1: <dig>  ready for ligation; 9: mixture of dna samples from lanes  <dig> and  <dig> in a molar ratio of 1: <dig>  after ligation; 10: mixture of dna samples from lanes  <dig> and  <dig> in a molar ratio of 6: <dig>  ready for ligation; 11: mixture of dna samples from lanes  <dig> and  <dig> in a molar ratio of 6: <dig>  after ligation; 12: dna ladder.
pcr products were purified using a qiaquick purification kit  and electrophoresed in 1% agarose with tris-acetate  as the running buffer
fig.
<dig> dna sequencing reveals that genes were correctly placed in the plasmid.
a upper panel: dna sequencing results of the forward cloning site of pet22b-rada; lower panel: dna sequencing results of the reverse cloning site of pet22b-rada.
b upper panel: dna sequencing results of the forward cloning site of pet22b-gene cluster 3; lower panel: dna sequencing results of the reverse cloning site of pet22b-gene cluster 3
long dna fragment cloning
to test the suitability of our method for a large dna fragment, we used it to insert a 20 kb dna fragment from the e. coli genome  containing  <dig> genes into pet22b between the start codon atg , and the sequence caccaccaccaccaccac  .
gene cluster  <dig> was amplified using the primer pair genecluster3-1/genecluster3- <dig>  as shown in additional file 4: table s <dig>  using e. coli genomic dna as template .
gene cluster  <dig> with a sticky end was generated by single-primer pcr, as shown in fig.
<dig>  using primer genecluster3- <dig> or genecluster3- <dig>  .
the ligation of gene cluster  <dig> dna fragment into pet22b was performed with t <dig> dna ligase using a molar ratio of vector to insert at 6: <dig>  as shown in fig.
<dig>  lane  <dig>  and  <dig> , insert and vector were ligated to one another and shifted to a higher molecular weight.
the inserted genes were amplified by colony pcr.
the presence of forward and reverse cloning sites were confirmed by dna sequencing .table  <dig> genes cloned and efficiency of reconstitution
yais
ybay
nfnb
yohk
rlmb
nhoa
dapa
deoc
deoa
deob
deod
yjjj
lpla
ytjb
serb
rada
yhdp
yjjk
slt
yjiq
yjir
yjis
yjit
yjiv
mcrc
mcrb
yjiw
hsds
hsdm
hsdr
mrr
yjia
recb
yjiy
tsr
yjiz
yjjm
yjjn
mdob
yeej
gast
mcm6
slc18a2
anumber of colonies checked
application of mrf cloning in genes reconstitution
in routine application of our cloning method, we created  <dig> constructs from e. coli genomic dna and human cdna  with inserts of variable size .
e. coli genes and gene clusters  were cloned from the e. coli genome.
the e. coli genes and gene clusters were subcloned into pet22b, with the gene under the control of a t <dig> promoter, and with a c-terminal his6-tag to facilitate protein purification .
human genes were subcloned into the expression vector pcdna™  <dig>    .
under our test conditions, we achieved an average cloning efficiency of  <dig> %.
dna sequencing revealed that all genes were correctly placed in the plasmid.
discussion
in this study, we describe a new cloning method.
the technique uses two rounds of pcr to obtain inserts and acceptors with compatible cohesive ends, which are then ligated.
using this method, we made  <dig> constructs with inserts of variable size.
the average cloning efficiency was  <dig> %, as determined by colony pcr and sequencing of the cloned genes.
because the method relies on pcr to generate cohesive 5’ or 3’ ends for dna ligation, restriction sites are not needed, which facilitates cloning of the gene of interest.
for convenience, we only used the vector pet22b for e. coli genes and pcdna™  <dig>   for human genes, but used inserts of variable size.
our results showed that cloning efficiency was not significantly affected by the different inserts, thus providing a glimpse of the wide choice in inserts that can be used as a template, which then can be used as an alternative method for multiple fragment assembly and library construction.
we noticed that cloning efficiency was not altered dramatically by fragment length.
as shown in table  <dig> and additional file 7: figure s <dig>  this method was suitable for the cloning of large dna sequences up to 20 kb in size.
in contrast to traditional restriction enzyme cloning, the method described here provides a much more flexible approach to gene cloning.
therefore, it represents a cost-effective and simple solution for high-throughput cloning applications.
because this method relies on pcr amplification of the dna sequences, the most crucial requirement is high-fidelity dna polymerase.
fortunately, the high-fidelity polymerases recently developed for cloning, e.g., phusion® high-fidelity dna polymerase and kod hot start dna polymerase, have extremely low error rates.
therefore, it is no longer challenging to amplify large dna fragments for use in our method.
CONCLUSIONS
we developed a novel cloning method that provides an alternative approach to dna assembly.
this method is independent of restriction sites and dpni treatment, and does not introduce undesired operational sequences at the junctions of functional modules.
this new method simplifies complex cloning procedures in which long stretches of dna can be inserted into circular plasmids in an unrestricted way, and the efficiency does not decrease for long inserts up to 20 kb.
the simplicity of both primer design and the procedure itself makes the method suitable for high-throughput studies.
the protein of interest is expressed without the addition of extra residues originating from the cloning procedure, making it an attractive alternative method for structural genomics.

 the manipulation of recombinant dna molecules is an indispensable step in modern high-throughput protein crystallization studies. restriction enzyme/ligase cloning, which relies on restriction enzyme digestion and ligation, is a simple and easy way to move a fragment of double-stranded dna from one plasmid to another. however, this technique has two limitations: it is ineffective when lack of unique restriction sites and sometimes results in introduction of unwanted extra sequences. to circumvent these limitations, various restriction endonuclease cleavage site–independent cloning methods have recently been developed. these methods have made cloning more accessible in cases in which conventional restriction site cloning was difficult or impossible. while as alternative cloning strategies are still required for more choices. the restriction-free cloning strategy, as described extensively in the literature, was developed as a powerful tool for reconstituting genes in circular vectors. because rf cloning requires no alterations in the plasmid or the gene of interest, it is exceptionally well-suited for high-throughput cloning. the gene of interest is amplified in a regular polymerase chain reaction, which produces a primer pair that, once annealed to the vector of interest, is extended in a linear amplification reaction. thus, this method relies on amplified genes functioning as primers. however, this approach also has limitations. first, the motion of large dna fragments and the formation of secondary structures will affect the efficiency of pcr. second, this method relies on digestion with dpni, which cleaves methylated dna, to remove parental plasmids. the efficiency of dpni treatment is influenced by many factors, and requires vector propagation in dam+ strains. in this paper, we describe a simple and fast method for performing gene reconstitution by modified restriction-free cloning. in this method, two rounds of pcr generate two dna fragments with compatible 5' or 3’ cohesive ends, which are therefore able to ligate to each other. this new method is independent of the existence of restrictions sites and dpni treatment. using this method, we made constructs with inserts of variable size, with average cloning efficiency higher than 85%. the efficiency was not significantly affected by the insert length up to 20 kb.  method overview and primer design figure  shows the scheme for mrf cloning. we define the 5–8 bp dna fragments before the insert site a as “5’ overhang” and those after insert site b as “3’ overhang”. to replace a gene in a vector between sites a and b, we designed eight primers: primer forward primer, which contains a ~25 bp sequence homologous to the positive strand of the gene; primer reverse primer, which contains a ~25 bp sequence homologous to the negative strand of the gene; primer forward primer, which is the same as primer but has an additional 5’ overhang at the 5’ end; primer reverse primer, which is the same as primer but has an additional 3’ overhang at the 5’ end; primer forward primer, which contains a ~25 bp sequence homologous to the negative strand of the vector; primer reverse primer, which contains a ~25 bp sequence homologous to the positive strand of the vector; primer reverse primer, which is the same as primer but has an additional 5' overhang at the 5’ end; primer reverse primer, which is the same as primer but has an additional 3’ overhang at the 5’ end. all primers were designed with g or c as the 5’ and 3’ terminal nucleotide.fig. schematic representation of mrf cloning. the insert gene or vector was amplified by regular double-primer pcr using genomic dna, cdna, or the original vector as template. compatible cohesive ends of insert gene or vector were created by two single-primer linear pcrs performed in parallel, followed by annealing of the two pcr products. inserts and acceptors with compatible cohesive ends were then assembled by ligation we used two pairs of primers, primer 1/primer and primer 5/primer to generate two dna fragments, using the target gene and vector as templates, respectively. the resultant pcr products were gel purified. we then amplified these two dna fragments in two separate pcr reactions containing either forward or reverse primer, which will add 5’ overhang or 3’ overhang to the pcr products. finally, dna fragments with complementary overhang at the 5’ or 3’ end were able to anneal to each other, and were joined by dna ligase. the ligated products were then transformed into dh5α competent cells. the inserted genes were verified by colony pcr and further confirmed by dna sequencing. mrf cloning can assemble insert dna fragment into target vector we first tested this protocol to reconstitute the e. coli rada gene into pet22b. based on the initial success of the protocol, we continued to employ it to generate the constructs needed for our studies. for example, we planned to replace the rada gene in vector pet22b between the start codon atg and the sequence caccaccaccaccaccac to yield a new construct with the gene under the control of a t promoter and a c-terminal his6-tag to facilitate protein purification. as shown in fig.  to replace the rada gene in the vector, two parallel pcrs were performed to amplify each dna fragment using the primer pairs pet22b1/pet22b and rada1/rada as shown in additional file 3: table s using pet22b or e. coli genomic dna as templates to generate dna fragments “1” and “2”. amplified products were separated by 1% agarose gel electrophoresis and purified by gel extraction. we then performed two separate single-primer linear pcrs: using pet22b or pet22b alone, with dna fragment “1” as the template, to obtain single-strand dna fragment “3-1” or “3-2”; and using rada or rada alone, with dna fragment “2” as template, to obtain single-strand dna fragment “4-1” or “4-2”. fragments “3-1” and “3-2” and “4-1” and “4-2” were then annealed to obtain double-strand dna fragments “3” and “4”, which have sticky ends that can ligate with other compatible ends. for single-primer pcr, we used ~500 ng of template, about times more than the standard amount recommended for double-primer pcr, as dna amplification in single-primer pcr is linear. as the efficiency of cohesive ligation is higher than that of blunt-end ligation, the parental dna of the products of second-round pcr did not need to be removed. after pcr purification, these second-round pcr products were ready for ligation. in each transformation, we routinely checked eight colonies at random from each transformation by colony pcr with a forward primer annealing to vector and a reverse primer annealing to the inserted gene.table pcr reaction components phusion high fidelity dna the agarose gel in fig.  shows the dna products of one sample at successive steps of our procedure. plasmid alone, prior to pcr, shows two major bands. after the double-primer pcr with forward and reverse primers, major bands can be seen at the expected sizes corresponding to the pcr-synthesized linear dna. single-primer pcr with forward or reverse yielded bands at the expected size of ~  kb and ~  kb after annealing the products of single-primer pcr, representing the pcr-synthesized linear dna with cohesive ends. additional smaller bands represent non-specific pcr products or single-strand dna. the ligation of insert into plasmid vector is performed by t dna ligase using a molar ratio of 1: vector to insert. as shown in fig.  lane and, insert and vector were ligated to one another and shifted to a higher molecular weight. the inserted genes were amplified by colony pcr. the presence of forward and reverse cloning sites were confirmed by dna sequencing.fig. gel electrophoresis separation of double-primer and single-primer pcr products. 1: parental plasmid pet22b alone; 2: pcr product from reaction with primers pet22b1/pet22b using plasmid pet22b as template; 3: pcr product from reaction with primers rada1/rada using e. coli genomic dna as template; 4: pcr product from reaction with primers genecluster3-1/genecluster3- using e. coli genomic dna as template; 5: annealed pcr products from two single-primer linear reactions using primer pet22b or pet22b and the dna sample from lane as template; 6: annealed pcr products from two single-primer linear reactions using the primer rada2fw or rada2rv, and the dna sample from lane as template; 7: annealed pcr products from two single-primer linear reactions using the primer genecluster3- or genecluster3- and the dna sample from lane as template; 8: mixture of dna samples from lanes and in a molar ratio of 1: ready for ligation; 9: mixture of dna samples from lanes and in a molar ratio of 1: after ligation; 10: mixture of dna samples from lanes and in a molar ratio of 6: ready for ligation; 11: mixture of dna samples from lanes and in a molar ratio of 6: after ligation; 12: dna ladder. pcr products were purified using a qiaquick purification kit and electrophoresed in 1% agarose with tris-acetate as the running buffer fig. dna sequencing reveals that genes were correctly placed in the plasmid. a upper panel: dna sequencing results of the forward cloning site of pet22b-rada; lower panel: dna sequencing results of the reverse cloning site of pet22b-rada. b upper panel: dna sequencing results of the forward cloning site of pet22b-gene cluster 3; lower panel: dna sequencing results of the reverse cloning site of pet22b-gene cluster 3 long dna fragment cloning to test the suitability of our method for a large dna fragment, we used it to insert a 20 kb dna fragment from the e. coli genome containing genes into pet22b between the start codon atg, and the sequence caccaccaccaccaccac. gene cluster was amplified using the primer pair genecluster3-1/genecluster3- as shown in additional file 4: table s using e. coli genomic dna as template. gene cluster with a sticky end was generated by single-primer pcr, as shown in fig.  using primer genecluster3- or genecluster3-. the ligation of gene cluster dna fragment into pet22b was performed with t dna ligase using a molar ratio of vector to insert at 6: as shown in fig.  lane and, insert and vector were ligated to one another and shifted to a higher molecular weight. the inserted genes were amplified by colony pcr. the presence of forward and reverse cloning sites were confirmed by dna sequencing.table genes cloned and efficiency of reconstitution yais ybay nfnb yohk rlmb nhoa dapa deoc deoa deob deod yjjj lpla ytjb serb rada yhdp yjjk slt yjiq yjir yjis yjit yjiv mcrc mcrb yjiw hsds hsdm hsdr mrr yjia recb yjiy tsr yjiz yjjm yjjn mdob yeej gast mcm6 slc18a2 anumber of colonies checked application of mrf cloning in genes reconstitution in routine application of our cloning method, we created constructs from e. coli genomic dna and human cdna with inserts of variable size. e. coli genes and gene clusters were cloned from the e. coli genome. the e. coli genes and gene clusters were subcloned into pet22b, with the gene under the control of a t promoter, and with a c-terminal his6-tag to facilitate protein purification. human genes were subcloned into the expression vector pcdna™. under our test conditions, we achieved an average cloning efficiency of %. dna sequencing revealed that all genes were correctly placed in the plasmid. discussion in this study, we describe a new cloning method. the technique uses two rounds of pcr to obtain inserts and acceptors with compatible cohesive ends, which are then ligated. using this method, we made constructs with inserts of variable size. the average cloning efficiency was %, as determined by colony pcr and sequencing of the cloned genes. because the method relies on pcr to generate cohesive 5’ or 3’ ends for dna ligation, restriction sites are not needed, which facilitates cloning of the gene of interest. for convenience, we only used the vector pet22b for e. coli genes and pcdna™ for human genes, but used inserts of variable size. our results showed that cloning efficiency was not significantly affected by the different inserts, thus providing a glimpse of the wide choice in inserts that can be used as a template, which then can be used as an alternative method for multiple fragment assembly and library construction. we noticed that cloning efficiency was not altered dramatically by fragment length. as shown in table  and additional file 7: figure s this method was suitable for the cloning of large dna sequences up to 20 kb in size. in contrast to traditional restriction enzyme cloning, the method described here provides a much more flexible approach to gene cloning. therefore, it represents a cost-effective and simple solution for high-throughput cloning applications. because this method relies on pcr amplification of the dna sequences, the most crucial requirement is high-fidelity dna polymerase. fortunately, the high-fidelity polymerases recently developed for cloning, e.g., phusion® high-fidelity dna polymerase and kod hot start dna polymerase, have extremely low error rates. therefore, it is no longer challenging to amplify large dna fragments for use in our method.  we developed a novel cloning method that provides an alternative approach to dna assembly. this method is independent of restriction sites and dpni treatment, and does not introduce undesired operational sequences at the junctions of functional modules. this new method simplifies complex cloning procedures in which long stretches of dna can be inserted into circular plasmids in an unrestricted way, and the efficiency does not decrease for long inserts up to 20 kb. the simplicity of both primer design and the procedure itself makes the method suitable for high-throughput studies. the protein of interest is expressed without the addition of extra residues originating from the cloning procedure, making it an attractive alternative method for structural genomics. 
restriction-free cloning, a pcr-based method for the creation of custom dna plasmids, allows for the insertion of any sequence into any plasmid vector at any desired position, independent of restriction sites and/or ligation. this new method does not require restriction sites or alterations of the plasmid or the gene of interest, or additional treatments. the amplified fragments were then used as the templates in two separate linear amplification reactions containing either forward or reverse primer to generate two single-strand reverse-complement counterparts, which could anneal to each other. here, we describe a simple and fast method for performing gene reconstitution by modified rf cloning. restriction-free cloninggene reconstitutionsingle-primer pcrhigh-throughput cloningagricultural university of hebeizd20162231301616zeng fanli hao zhimin http://dx.doi.org/ /501100008525agricultural university of hebei31271997dong jingao issue-copyright-statement© the author 2017 the simplicity of both primer design and the procedure itself makes the method suitable for high-throughput cloning and structural genomics. the average cloning efficiency was higher than 85%, as confirmed by colony pcr and sequencing of the inserts. the annealed inserts and acceptors with 5’ and 3’ cohesive ends were sealed by ligation reaction. our method provides an alternative cloning method capable of inserting any dna fragment of up to at least 20 kb into a plasmid, with high efficiency.
 restriction-free cloning, a pcr-based method for the creation of custom dna plasmids, allows for the insertion of any sequence into any plasmid vector at any desired position, independent of restriction sites and/or ligation. here, we describe a simple and fast method for performing gene reconstitution by modified rf cloning.  double-stranded inserts and acceptors were first amplified by regular pcr. the amplified fragments were then used as the templates in two separate linear amplification reactions containing either forward or reverse primer to generate two single-strand reverse-complement counterparts, which could anneal to each other. the annealed inserts and acceptors with 5’ and 3’ cohesive ends were sealed by ligation reaction. using this method, we made constructs containing insertions of up to 20 kb. the average cloning efficiency was higher than 85%, as confirmed by colony pcr and sequencing of the inserts.  our method provides an alternative cloning method capable of inserting any dna fragment of up to at least 20 kb into a plasmid, with high efficiency. this new method does not require restriction sites or alterations of the plasmid or the gene of interest, or additional treatments. the simplicity of both primer design and the procedure itself makes the method suitable for high-throughput cloning and structural genomics. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords restriction-free cloninggene reconstitutionsingle-primer pcrhigh-throughput cloningagricultural university of hebeizd20162231301616zeng fanli hao zhimin http://dx.doi.org/ /501100008525agricultural university of hebei31271997dong jingao issue-copyright-statement© the author 2017 