Two new techniques were employed to develop a new approach to customize DNA sequences. The first technique uses a new group of nicking endonucleases, which cleave only one strand of ds DNA in a site-specific manner (22–24
). This site-specific nicking is used to create insertion sites in vectors with unique non-palindromic 3′ ss extensions. Ideally, the vector backbone should not have additional nicking sites besides those in the cassette. Thus, to create pNEB206A vector we used the nicking endonuclease Nt.BbvCI (24
) which recognizes the seven base pair sequence, CC↓TGAGG, and which is not present (or infrequently present) in the sequences of commonly used cloning vectors. We have now determined that a complete absence of additional nicking sites is not essential as long as nicked sites on opposite strands are separated by at least 100
bp. Currently, a variety of site-specific nicking endonucleases recognizing either six or seven base pair sequences are commercially available from New England Biolabs, therefore the design of single-stranded extensions on the vectors may be custom modified by replacing the nicking/restriction sites in the cassette. Recently, a comprehensive set of the USER compatible vectors has been constructed by other researchers for constitutive expression in a variety of host organisms (35
The other technique employed to customize DNA sequences is an improvement to a ‘ligase-free UDG cloning’ method (11
). DNA amplification using primers with a single dU placed near their 5′ end followed by incubation with the USER enzyme creates PCR fragments with unique non-palindromic 3′ ss extensions. This allows for accurate, multi-fragment assemblies among the multiple PCR fragments, as well as insertion into the vector, all in one step.
The concept and experiments presented here are flexible, in that, many different nicking enzyme specificities could be used. Furthermore, different modified bases along with their cognate ‘repair’ enzyme can be used to create breaks near the 5′ ends of amplified fragments. Several E. coli
DNA glycosylases/AP lyases are capable of breaking phosphodiester bonds at the AP-sites (19
). For example, formamidopyrimidine-DNA glycosylase, also known as FPG, can excise a variety of oxidized purines (29
), whereas either DNA endonucleasae III or endonuclease VIII both can excise a variety of oxidized pyrimidines (19
). Due to the AP-lyase activity, which can act independently of associated glycosylase activity, these enzymes are also capable of breaking phosphodiester bonds at AP sites, but Endo III breaks the first phosphodiester bond 3′ to the AP site (28
), whereas FPG and EndoVIII are capable of breaking phosphodiester bonds on both 3′ and 5′ sides of the AP site (19
). In general, the combination of DNA glycosylases in the mixture depends on (i) the type of modified nucleotide to be excised and (ii) the type of termini desired at the nick location. For example, by combining the uracil DNA glycosylase hSMUG1 (30
) with EndoVIII we have created a nicking tool, which is specific for hydroxymethyluracil (data not shown). A combination of three enzymes, UDG, EndoIV and EndoVIII, was used to nick at dU and generate a 5′ phosphate and 3′ hydroxyl at DNA termini (data not shown).
We created an efficient and fast method for assembly of desired DNA molecules from multiple PCR fragments, referred to as the USER friendly DNA engineering method. The most distinctive feature of the technique is its universality, as it combines nucleotide sequence manipulation, PCR fragment assembly and directional cloning in a single experimental format. By modifications in the primers, which, during DNA amplification are incorporated into the ends of the PCR products, virtually any envisioned DNA manipulation may be performed at any location of a target DNA. The junction of PCR fragments across single-stranded extensions generated by the USER enzyme is extremely precise. Since the assembly reaction is ligase free, the extensions with mismatched sequences do not yield stable recombinant molecules and are lost during transformation. In addition, when proofreading polymerase PfuCx
is used for DNA amplification, the PCR errors are rare. PfuCx
DNA polymerase is a genetically engineered derivative of Pfu
). No wild-type proofreading DNA polymerases, such as Vent
, can yield PCR product from primers carrying dU (14
). Archaeal DNA polymerases possess a uracil-binding pocket which blocks extension past a uridine (20
). In addition to PfuCx
, the other DNA polymerase compatible with the USER friendly DNA engineering method is Taq
DNA polymerase. Despite the fact that Taq
polymerase has a higher error rate, we have successfully used it in numerous site-specific mutagenesis and gene fusion experiments where a two or three PCR fragment assembly was required.
To date, the highest number of PCR fragments successfully assembled in one reaction was seven. It should be pointed out that the efficiency of directional assembly of PCR fragments to a great degree depends on the uniqueness of nucleotide sequences at the junctions. However, in some cases, the choices for junction sequence selection are very limited due to unfavorable A/T content of the target DNA. Thus, when more than five PCR fragments are assembled in the same reaction, there is a greater possibility that junctions will have similar sequences and the fragments may tend to assemble incorrectly, which significantly reduces the number of recombinants. For example, in one of our experiments the goal was to delete seven restriction sites from the LexA gene, thus a six-fragment assembly was performed to introduce seven silent mutations simultaneously. Initially, the percentage of recombinants was very poor (14%), but by shifting the position of one faulty junction we were able to eliminate the mis-annealing of two intermediate fragments and the yield of recombinants was increased to 60%.
In addition to altering the sequence of a target DNA, the USER enzyme could be used in conjunction with PfuCx DNA polymerase to amplify the cloning vector and in the same reaction add sequence junctions compatible for assembly of targeted PCR fragments. This approach was successfully employed to generate precise protein fusions for rapid protein purification using either the IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) system or the pMAL™ Protein Fusion and Purification system (31,32). In conclusion, the rapidity, precision and efficacy of the USER friendly DNA engineering method outweigh the higher costs of dU in PCR primers, as well as a limited choice of DNA polymerases.