BACKGROUND
uracil-excision-based cloning was invented more than  <dig> years ago  <cit> , but the technique was left unused, due to incompatibility with high-fidelity pcr  <cit> .
in the technique, compatible single stranded dna overhangs are created by substituting selected deoxy thymidine  nucleotides with deoxy uridine nucleotides  .
subsequently, the dna is treated with uracil dna glycosidase  and usually either t <dig> endonuclease  <cit>  or dna glycosylase-lyase endo viii , which releases the sequence upstream from the du's and allows pairing between exposed, compatible ends .
the overhangs are usually designed to be 7- <dig> nucleotides long, and therefore can create circular dna species that are stable enough to allow bacterial transformation without prior ligation.
hence, the technique relies solely on a pair of properly spaced deoxy adenine- and dt-nucleotides, and, due to the degeneracy of the genetic code, allow seamless translational fusions of virtually any protein coding dna sequence, making it particular suitable for  applications such as chimeric dna fusion designs.
in  <dig>  the crystal structure of the dna polymerase tgo from the archea thermococcus gorgonarius was solved  <cit> , revealing the nature of the uracil-binding pocket, and allowing the design of mutant tgo- and pfu-polymerases with reduced stalling at uracil-containing dna  <cit> .
with mutants like pfuv93q, high-fidelity pcr became compatible with uracil-excision cloning.
this led to development of an improved uracil-excision cloning technology  <cit>  and to a new way of doing seamless pcr product fusion that may eventually replace overlapping pcr in many applications  <cit> .
the useful application of these technologies was further demonstrated by their use in artificial gene synthesis  <cit> .
in addition to pcr, and cloning and fusing genes, site-directed mutagenesis is an indispensable tool for molecular biologists.
one of the early methods for doing site-directed mutagenesis was the kunkel method  <cit>  that uses template dna isolated from a ung- dut- e. coli strain.
this strain lacks dutpase and uracil deglycosidase and therefore accumulates soluble dutp and dna-bound du nucleotides.
in the method, du-containing dna is used as template in a linear amplification reaction with a mutagenic primer, as well as the klenow enzyme, dntps and a ligase.
subsequently, the dna is transformed into ung+ bacteria, where only the newly synthesized, mutant dna survives and the primer-introduced mutation is isolated.
later, pcr entered the scene and variants, known as inverse pcr or whole plasmid synthesis , largely seems to have replaced the kunkel method.
in the typical pcr-based approach, template carry-over is inhibited by treatment with the restriction enzyme dpni, that restricts dam methylated plasmid dna, but leaves unmethylated pcr-derived dna intact.
uracil-excision can be used in several ways as a simple, versatile and large-insertion compatible site-mutagenesis whops method.
the method offers several advantages compared to previous whops protocols .
first, whereas traditional exponential amplification whops uses two unique primers for each mutation , uracil-excision offers the opportunity to reuse one primer when several mutations need to be introduced in one location .
second, the primers do not to overlap in their 3'-end, and, hence, are shorter and should perform better in pcr .
third, given the limitation in dna-synthesis chemistry, the reduced size of the primers offers the opportunity to create larger insertion mutations  and offers the same possibilities with deletions .
fourth, uracil-excision can be used for very large insertions , free from the inherent limitations in overlapping pcr.
however, most importantly, the protocol is highly versatile and insertions can be created from a variety of dna species, ranging from small, perfectly complementary dna oligonucleotides, over medium-range dna species created by enzymatic extension of oligonucleotides that only overlap in their 3'end, to dna of virtually any length created by pcr - and several different approaches can be combined in one-tube format to introduce various changes in one reaction.
here, uracil-excision-based artificial gene synthesis is used to create a combination of a pfuv93q mutant  <cit>  and a highly processive pfu-sso7d fusion polymerase  <cit> .
this new dna-polymerase has several properties that make it uniquely suitable for several of the described applications, including site-directed mutagenesis of large plasmids, and a combination of the modern whops mutagenesis and the classical kunkel-method to avoid carry-over of template dna.
RESULTS
fusing the dna-binding protein sso7d to pfuv93q yields a highly efficient polymerase for use in uracil-excision dna engineering
one of the drawbacks of uracil-excision molecular cloning and pcr fusion, is that it is only compatible with two different, commercially available dna polymerases: the non-proof-reading taq polymerase  and the proof-reading polymerase pfucx , both which suffer from having low processivity  <cit> .
recently, the processivity of both taq and pfu was greatly improved by fusing the enzymes to the small dna-binding protein sso7d , derived from the thermophilic archea sulfolobus solfataricus  <cit> .
in our lab, it has been found that the commercial version of the pfu fusion protein , performs significantly better in whops of large  plasmids.
hence, the uracil-binding pocket mutation  version of pfus <dig> will greatly improve the performance of the uracil-excision toolkit, in particular in challenging pcr reactions such as site-directed mutagenesis on large plasmids.
a v93q version of pfu was first created in a pet-expression vector system by standard whops.
subsequently, due to the lack of an sso7d dna template, a uracil-excision based artificial gene synthesis strategy was employed to add an artificial sso7d gene to the 3'end of the pfuv93q expression construct .
this example demonstrates that uracil-excision technology can be used to create entirely artificial genes and this approach is compatible in one-tube format with the other uracil-excision applications such as pcr fragment cloning and site-directed mutagenesis, expanding the versatility of the technology.
using standard pfu polymerase conditions, we next compared the performance of the purified pfuv93q-s <dig> enzyme  with the commercial pfuturbo , pfus <dig> , the commercial pfuv93q-turbo , and pfuv93q using both standard and uracil-containing primers, in a challenging mutagenesis reaction, on a large  e. coli-yeast shuttle vector ptef <dig>  <cit>  harboring the plant gene cre <dig>  <cit> .
the two nearly identical primer pairs were designed to fit either a uracil-excision strategy or a very similar whops strategy  <cit> , and used to introduce a total of  <dig> mutations in  <dig> neighboring codons in the cre <dig> sequence.
standard 1% agarose gel electrophoresis analysis of the products showed that the two sso7d fusion polymerases were able to amplify the plasmid with standard primers, whereas only pfux <dig> amplified the plasmid with uracil-containing primers .
various conditions were tried, including cycling conditions with very long extension times, but it was never possible to amplify the plasmid with a non-sso7d polymerase.
pfux <dig> polymerase is compatible with the kunkel-method to limit carry-over of unwanted template dna
previously, two methods have been used to limit template carry-over in site-directed mutagenesis reactions.
in the classical kunkel-method, the template is isolated from a ung- dut- strain of e. coli such as ch <dig> , which makes it incompatible with re-transformation into normal ung+ bacteria.
exponential whops mutagenesis takes advantage of the dpni restriction enzyme to restrict methylated template dna.
the combined use of du-containing dna with dpni-restriction was previously demonstrated with a linear amplification mutagenesis strategy  <cit> , but the pfuv93q versions allow pcr-based site-directed mutagenesis to be combined with both template elimination methods and hence, will greatly decrease the recovery of unwanted template dna.
a simple screen was designed to test this idea, based on a single nucleotide mutation of the green fluorescent protein turning the codon coding for tyrosine  <dig> into a tag stop codon.
in the screen, mutants are easily identified as non-fluorescent colonies .
a gfp expression vector  <cit>  was isolated from both cj <dig> and the common ung+ cloning strain dh5α and used as template in a common 13-cycle protocol with pfu-turbo or pfux <dig>  as expected, only pfux <dig> was able to amplify dna isolated from the cj <dig> strain .
similar results were obtained comparing pfux <dig> to phusion/pfus <dig> polymerase .
next, aliquots of the reactions were treated with dpni and the dpni-treated and untreated reactions were transformed into the bl <dig> strain.
the screen confirmed that very few template-containing colonies were isolated from reactions based on dna isolated from cj <dig>  whereas a large proportion of the colonies formed from the dh5α-dna based reactions exhibited the wildtype, fluorescent phenotype .
quantifications of the results showed that in all cases, using dna from cj <dig> or dpni-treatment, resulted in more than 90% mutant recovery .
pfux <dig> allows the use of dutp instead of dttp in pcr
pcr revolutionized molecular cloning and largely defined the field of forensic genetics and allowed the analysis of ancient dna samples of low quality and quantity.
however, the efficiency of pcr creates a risk of contamination with previous samples and is of particular concern in e.g.
forensic genetics.
several methods have been developed to prevent pcr carry-over contamination.
in a widely used method, dutp replaces dttp in pcr, and prior to a new reaction ung-treatment destroys all du-containing, contaminating dna species  <cit> .
pfux <dig> accepts the replacement of dttp with dutp in pcr, and therefore is compatible with ung-based carry over contamination prevention.
this was demonstrated with a standard pcr, with or without dttp replaced by dutp.
in the experiment, pfux <dig> was compared to the other highly processive phusion polymerase using standard phusion pcr conditions.
agarose gel electrophoresis confirmed that only pfux <dig> was able to perform pcr with dutp instead of dttp .
discussion
in one of the early pcr-based mutagenesis protocols, whops was performed with non-overlapping phosphorylated primers and was followed by a ligation step prior to transformation of the mutant dna  <cit>  - the method became available as the exsite™ pcr-based site-directed mutagenesis kit  and a similar product is today available as the phusion™ site-directed mutagenesis kit .
the uracil-excision site-directed mutagenesis approach is very similar.
however, compared to the latter technique, ligation is not necessary and the uracil-defined overlaps add an extra quality insurance step since erroneous mismatching extensions on the amplified plasmid will not yield stable recombinant molecules  <cit> .
in the older approach, any phosphorylated dna species can be ligated to create a circular recombinant molecule.
today, probably the most widely used technique is whops with a perfectly overlapping primer pair that negates the need for ligation prior to transformation - commercially available from e.g.
stratagene as the quikchange® site-directed mutagenesis kit.
due to the exponential nature of whops, and the availability of modern high-fidelity dna polymerases, such as the highly processive pfu-sso7d fusion protein dna polymerase  <cit> , this is a highly efficient technology, but the technique is not as versatile as uracil-excision.
in site-directed mutagenesis, the dpni template removal step adds extra time to the procedure and template carry-over is an error frequently observed in our lab.
pfux <dig> allows the combined use of dpni-treatment and du-containing dna to avoid carry-over of template.
in our experimental setup, both the kunkel-method and the use of dpni was more than 90% efficient in the prevention of template recovery, and it was therefore not possible to detect a large effect of the combined use.
however, in a linear amplification mutagenesis strategy, the combined use of dpni and du-dna was previously reported to increase the efficiency of a mutagenesis from 38%  to 70-91%  <cit> .
therefore, the kunkel-approach to limit tempate carry-over could be a useful and time-saving alternative or addition to dpni-treatment, in site-directed mutagenesis.
pfu-sso7d fusion polymerases, such as phusion, are marketed as polymerases for direct pcr on complex samples such as blood.
indeed, we have found that in some cases pfux <dig> is the only dna polymerase that produces a pcr product in combination with dna isolated from e.g.
plant material , human cdna  or when doing whops on large plasmid dna templates .
forensic pcr deals with complex samples of low quantity and quality, and prevention of carry-over contamination from previous pcrs is important to this field.
pfux <dig> polymerase performs better than other dna polymerases in virtually all applications in our laboratory.
furthermore, none of the wildtype pfu-based versions  have ever been able to amplify du-containing dna.
hence, pfux <dig> polymerase may be useful in forensic pcr, both due to its high performance as well as compatibility with the well-established ung-method for prevention of carry-over contamination.
CONCLUSIONS
the combination of the user enzyme and the new pfux <dig> polymerase described here, constitutes a simple, yet comprehensive molecular toolkit that enables one-tube, ligase-free cloning  <cit> , easy conversion or design of compatible vectors  <cit> , seamless pcr fusions  <cit> , artificial gene synthesis  <cit> , site-directed mutagenesis and prevention of carry-over contamination.
no other single molecular cloning technology exhibits a comparable versatility.

 uracil-excision-based cloning was invented more than years ago, but the technique was left unused, due to incompatibility with high-fidelity pcr. in the technique, compatible single stranded dna overhangs are created by substituting selected deoxy thymidine nucleotides with deoxy uridine nucleotides. subsequently, the dna is treated with uracil dna glycosidase and usually either t endonuclease or dna glycosylase-lyase endo viii, which releases the sequence upstream from the du's and allows pairing between exposed, compatible ends. the overhangs are usually designed to be 7- nucleotides long, and therefore can create circular dna species that are stable enough to allow bacterial transformation without prior ligation. hence, the technique relies solely on a pair of properly spaced deoxy adenine- and dt-nucleotides, and, due to the degeneracy of the genetic code, allow seamless translational fusions of virtually any protein coding dna sequence, making it particular suitable for applications such as chimeric dna fusion designs. in the crystal structure of the dna polymerase tgo from the archea thermococcus gorgonarius was solved, revealing the nature of the uracil-binding pocket, and allowing the design of mutant tgo- and pfu-polymerases with reduced stalling at uracil-containing dna. with mutants like pfuv93q, high-fidelity pcr became compatible with uracil-excision cloning. this led to development of an improved uracil-excision cloning technology and to a new way of doing seamless pcr product fusion that may eventually replace overlapping pcr in many applications. the useful application of these technologies was further demonstrated by their use in artificial gene synthesis. in addition to pcr, and cloning and fusing genes, site-directed mutagenesis is an indispensable tool for molecular biologists. one of the early methods for doing site-directed mutagenesis was the kunkel method that uses template dna isolated from a ung- dut- e. coli strain. this strain lacks dutpase and uracil deglycosidase and therefore accumulates soluble dutp and dna-bound du nucleotides. in the method, du-containing dna is used as template in a linear amplification reaction with a mutagenic primer, as well as the klenow enzyme, dntps and a ligase. subsequently, the dna is transformed into ung+ bacteria, where only the newly synthesized, mutant dna survives and the primer-introduced mutation is isolated. later, pcr entered the scene and variants, known as inverse pcr or whole plasmid synthesis, largely seems to have replaced the kunkel method. in the typical pcr-based approach, template carry-over is inhibited by treatment with the restriction enzyme dpni, that restricts dam methylated plasmid dna, but leaves unmethylated pcr-derived dna intact. uracil-excision can be used in several ways as a simple, versatile and large-insertion compatible site-mutagenesis whops method. the method offers several advantages compared to previous whops protocols. first, whereas traditional exponential amplification whops uses two unique primers for each mutation, uracil-excision offers the opportunity to reuse one primer when several mutations need to be introduced in one location. second, the primers do not to overlap in their 3'-end, and, hence, are shorter and should perform better in pcr. third, given the limitation in dna-synthesis chemistry, the reduced size of the primers offers the opportunity to create larger insertion mutations and offers the same possibilities with deletions. fourth, uracil-excision can be used for very large insertions, free from the inherent limitations in overlapping pcr. however, most importantly, the protocol is highly versatile and insertions can be created from a variety of dna species, ranging from small, perfectly complementary dna oligonucleotides, over medium-range dna species created by enzymatic extension of oligonucleotides that only overlap in their 3'end, to dna of virtually any length created by pcr - and several different approaches can be combined in one-tube format to introduce various changes in one reaction. here, uracil-excision-based artificial gene synthesis is used to create a combination of a pfuv93q mutant and a highly processive pfu-sso7d fusion polymerase. this new dna-polymerase has several properties that make it uniquely suitable for several of the described applications, including site-directed mutagenesis of large plasmids, and a combination of the modern whops mutagenesis and the classical kunkel-method to avoid carry-over of template dna.  fusing the dna-binding protein sso7d to pfuv93q yields a highly efficient polymerase for use in uracil-excision dna engineering one of the drawbacks of uracil-excision molecular cloning and pcr fusion, is that it is only compatible with two different, commercially available dna polymerases: the non-proof-reading taq polymerase and the proof-reading polymerase pfucx, both which suffer from having low processivity. recently, the processivity of both taq and pfu was greatly improved by fusing the enzymes to the small dna-binding protein sso7d, derived from the thermophilic archea sulfolobus solfataricus. in our lab, it has been found that the commercial version of the pfu fusion protein, performs significantly better in whops of large plasmids. hence, the uracil-binding pocket mutation version of pfus will greatly improve the performance of the uracil-excision toolkit, in particular in challenging pcr reactions such as site-directed mutagenesis on large plasmids. a v93q version of pfu was first created in a pet-expression vector system by standard whops. subsequently, due to the lack of an sso7d dna template, a uracil-excision based artificial gene synthesis strategy was employed to add an artificial sso7d gene to the 3'end of the pfuv93q expression construct. this example demonstrates that uracil-excision technology can be used to create entirely artificial genes and this approach is compatible in one-tube format with the other uracil-excision applications such as pcr fragment cloning and site-directed mutagenesis, expanding the versatility of the technology. using standard pfu polymerase conditions, we next compared the performance of the purified pfuv93q-s enzyme with the commercial pfuturbo, pfus, the commercial pfuv93q-turbo, and pfuv93q using both standard and uracil-containing primers, in a challenging mutagenesis reaction, on a large e. coli-yeast shuttle vector ptef harboring the plant gene cre. the two nearly identical primer pairs were designed to fit either a uracil-excision strategy or a very similar whops strategy, and used to introduce a total of mutations in neighboring codons in the cre sequence. standard 1% agarose gel electrophoresis analysis of the products showed that the two sso7d fusion polymerases were able to amplify the plasmid with standard primers, whereas only pfux amplified the plasmid with uracil-containing primers. various conditions were tried, including cycling conditions with very long extension times, but it was never possible to amplify the plasmid with a non-sso7d polymerase. pfux polymerase is compatible with the kunkel-method to limit carry-over of unwanted template dna previously, two methods have been used to limit template carry-over in site-directed mutagenesis reactions. in the classical kunkel-method, the template is isolated from a ung- dut- strain of e. coli such as ch, which makes it incompatible with re-transformation into normal ung+ bacteria. exponential whops mutagenesis takes advantage of the dpni restriction enzyme to restrict methylated template dna. the combined use of du-containing dna with dpni-restriction was previously demonstrated with a linear amplification mutagenesis strategy, but the pfuv93q versions allow pcr-based site-directed mutagenesis to be combined with both template elimination methods and hence, will greatly decrease the recovery of unwanted template dna. a simple screen was designed to test this idea, based on a single nucleotide mutation of the green fluorescent protein turning the codon coding for tyrosine into a tag stop codon. in the screen, mutants are easily identified as non-fluorescent colonies. a gfp expression vector was isolated from both cj and the common ung+ cloning strain dh5α and used as template in a common 13-cycle protocol with pfu-turbo or pfux as expected, only pfux was able to amplify dna isolated from the cj strain. similar results were obtained comparing pfux to phusion/pfus polymerase. next, aliquots of the reactions were treated with dpni and the dpni-treated and untreated reactions were transformed into the bl strain. the screen confirmed that very few template-containing colonies were isolated from reactions based on dna isolated from cj whereas a large proportion of the colonies formed from the dh5α-dna based reactions exhibited the wildtype, fluorescent phenotype. quantifications of the results showed that in all cases, using dna from cj or dpni-treatment, resulted in more than 90% mutant recovery. pfux allows the use of dutp instead of dttp in pcr pcr revolutionized molecular cloning and largely defined the field of forensic genetics and allowed the analysis of ancient dna samples of low quality and quantity. however, the efficiency of pcr creates a risk of contamination with previous samples and is of particular concern in e.g. forensic genetics. several methods have been developed to prevent pcr carry-over contamination. in a widely used method, dutp replaces dttp in pcr, and prior to a new reaction ung-treatment destroys all du-containing, contaminating dna species. pfux accepts the replacement of dttp with dutp in pcr, and therefore is compatible with ung-based carry over contamination prevention. this was demonstrated with a standard pcr, with or without dttp replaced by dutp. in the experiment, pfux was compared to the other highly processive phusion polymerase using standard phusion pcr conditions. agarose gel electrophoresis confirmed that only pfux was able to perform pcr with dutp instead of dttp. discussion in one of the early pcr-based mutagenesis protocols, whops was performed with non-overlapping phosphorylated primers and was followed by a ligation step prior to transformation of the mutant dna - the method became available as the exsite™ pcr-based site-directed mutagenesis kit and a similar product is today available as the phusion™ site-directed mutagenesis kit. the uracil-excision site-directed mutagenesis approach is very similar. however, compared to the latter technique, ligation is not necessary and the uracil-defined overlaps add an extra quality insurance step since erroneous mismatching extensions on the amplified plasmid will not yield stable recombinant molecules. in the older approach, any phosphorylated dna species can be ligated to create a circular recombinant molecule. today, probably the most widely used technique is whops with a perfectly overlapping primer pair that negates the need for ligation prior to transformation - commercially available from e.g. stratagene as the quikchange® site-directed mutagenesis kit. due to the exponential nature of whops, and the availability of modern high-fidelity dna polymerases, such as the highly processive pfu-sso7d fusion protein dna polymerase, this is a highly efficient technology, but the technique is not as versatile as uracil-excision. in site-directed mutagenesis, the dpni template removal step adds extra time to the procedure and template carry-over is an error frequently observed in our lab. pfux allows the combined use of dpni-treatment and du-containing dna to avoid carry-over of template. in our experimental setup, both the kunkel-method and the use of dpni was more than 90% efficient in the prevention of template recovery, and it was therefore not possible to detect a large effect of the combined use. however, in a linear amplification mutagenesis strategy, the combined use of dpni and du-dna was previously reported to increase the efficiency of a mutagenesis from 38% to 70-91%. therefore, the kunkel-approach to limit tempate carry-over could be a useful and time-saving alternative or addition to dpni-treatment, in site-directed mutagenesis. pfu-sso7d fusion polymerases, such as phusion, are marketed as polymerases for direct pcr on complex samples such as blood. indeed, we have found that in some cases pfux is the only dna polymerase that produces a pcr product in combination with dna isolated from e.g. plant material, human cdna or when doing whops on large plasmid dna templates. forensic pcr deals with complex samples of low quantity and quality, and prevention of carry-over contamination from previous pcrs is important to this field. pfux polymerase performs better than other dna polymerases in virtually all applications in our laboratory. furthermore, none of the wildtype pfu-based versions have ever been able to amplify du-containing dna. hence, pfux polymerase may be useful in forensic pcr, both due to its high performance as well as compatibility with the well-established ung-method for prevention of carry-over contamination.  the combination of the user enzyme and the new pfux polymerase described here, constitutes a simple, yet comprehensive molecular toolkit that enables one-tube, ligase-free cloning, easy conversion or design of compatible vectors, seamless pcr fusions, artificial gene synthesis, site-directed mutagenesis and prevention of carry-over contamination. no other single molecular cloning technology exhibits a comparable versatility. 
here, the addition of a highly efficient dna polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision dna engineering.
 the combined use of restriction enzymes with pcr has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated dna sequences. uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple dna sequences in simple one-tube reactions, with higher accuracy than overlapping pcr.  here, the addition of a highly efficient dna polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision dna engineering.  the different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning. 