A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

Background: Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable. Results: We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange (TM) site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis. Conclusion: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange (TM). Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange T) but increased the overall success rates.Publisher PDFPeer reviewe

Investigating thermostable DNA polymerases for PCR-based applications

PhD ThesisThermostable DNA polymerases are essential components of the polymerase chain reaction (PCR), a technique widely applied across the entire biosciences. The work presented in this thesis improves understanding of the function and properties of these enzymes with the aim of developing improved formulations for biotechnological applications. The accuracy with which polymerases replicate DNA is essential for their application in PCR. A plasmid-based DNA polymerase fidelity assay, based on a gapped plasmid template containing the lacZα gene, has been developed. This technique, a marked improvement on previous methods, enables straightforward determination of any polymerase’s fidelity. The functions of two loops in archaeal family-B DNA polymerases, located in the thumb domain responsible for double-stranded DNA binding, have been elucidated, revealing a role in the control of polymerase and proof-reading exonuclease activities. Site-directed mutagenesis, combined with kinetic and binding experiments, was used for this purpose. The family-B DNA polymerase from the archaeon Pyrococcus furiosus has low processivity, limiting its ability to amplify long stretches of DNA. The processivity of this enzyme was increased by changing a number of amino acids to those observed in the more processive polymerase from Thermococcus kodakarensis. Several mutants have been identified with increased processivity and improved performance in PCR. Reverse transcription PCR (RT-PCR) typically requires the use of a mesophilic reverse transcriptase to generate cDNA from RNA, which is then amplified by a thermostable DNA polymerase in PCR. Through the use of compartmentalised self-replication (CSR) and rational design, generation of a DNA polymerase with reverse transcriptase activity capable of single tube RT-PCR was attempted

simple version of megaprimer pcr for site directed mutagenesis

n/

Simultaneous splicing of multiple DNA fragments in one PCR reaction

BACKGROUND: Rapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects. However, former overlap extension PCRs, the most common methods for splicing DNA fragments, are not really simultaneous fusing of multiple DNA fragments. RESULTS: We performed an optimized method which allowed simultaneous splicing of multiple DNA fragments in one PCR reaction. Shorter outermost primers were prior mixed with other PCR components at the same time. A sequential thermo cycling program was adopted for overlap extension reaction and amplification of spliced DNA. Annealing temperature was relatively higher in the overlap extension reaction stage than in the fused DNA amplification. Finally we successfully harvested target PCR products deriving from fusion of two to seven DNA fragments after 5–10 cycles for overlap extension reaction and then 30 cycles for fused DNA amplification. CONCLUSIONS: Our method provides more rapid, economical and handy approach to accurately splice multiple DNA fragments. We believe that our simultaneous splicing overlap extension PCR can be used to fuse more than seven DNA fragments as long as the DNA polymerase can match

Fidelity of eukaryotic and archaeal family-B DNA polymerases

PhD ThesisDNA polymerases are essential for replication, recombination and repair of DNA. However these enzymes have multiple applications in biotechnology. Since PCR has been developed, thermostable DNA polymerases have become important for numerous PCR based applications. Currently these enzymes are used routinely in laboratories all over the world. The fidelity of DNA polymerases is a key feature for PCR. We have developed a fidelity assay, based on a gapped plasmid template containing the lacZα gene reporter, allowing easy and straightforward measurement of the accuracy of DNA synthesis by DNA polymerases in vitro. Previous studies on the family-B DNA polymerase from Pyrococcus furiosus demonstrated that fidelity is controlled by D473, an amino acid located in the loop of the fingers domain. It was observed that the mutation D473G had a strong error-prone phenotype and Pfu-Pol D473G can be successfully used for random mutagenesis. To test if eukaryotic family-B DNA polymerases use the same aspartic acid residue to control fidelity we prepared the D799G mutant of a proteolytic fragment of polymerase epsilon from Saccharomyces cerevisiae. Unfortunately we did not observe the expected modulation of the fidelity of DNA synthesis for the D799G polymerase epsilon variant. Overexpression of the multi-subunit family-B DNA polymerases from Saccharomyces cerevisiae was found to be extremely demanding. Therefore, we decided to modify the thermostable family-B DNA polymerase from Thermococcus gorgonarius to obtain variants containing the loop region of the fingers domain from family-B DNA polymerases of Saccharomyces cerevisiae. We have observed no change in DNA synthesis accuracy when the loop region was transferred from high fidelity yeast replicative polymerase delta. However when the loop region was transferred from Saccharomyces cerevisiae error-prone family-B DNA polymerase zeta we observed a strong error-prone phenotype, in some instances, loop swapping with polymerase zeta is complicated by alignment ambiguity, so several variants were prepared. The primary sequence alignment of the fingers domain of eukaryotic polymerases zeta suggests no strong consensus within the loop region. Therefore, we decided to replace the major part of the fingers domain of the family-B polymerase from Thermococcus gorgonarius with the equivalent functional module from Saccharomyces cerevisiae polymerase zeta. The main aim of such a rearrangement was to test if the module from the error-prone DNA polymerase zeta has the potential to decrease fidelity. The chimeric polymerase variant was indeed found to be a very inaccurate DNA polymerase. To our surprise we also discovered that the polymerase variant possesses reverse transcriptase activity. Several further modifications allowed us to significantly improve reverse transcriptase activity of the chimeric polymerase variant

Thermostable DNA polymerases in replication, repair and biotechnology

PhD ThesisMany archaea contain a unique DNA polymerase, DNA Pol D. This enzyme is a heterodimer composed of a large subunit (polymerase) and a small subunit (3’- 5’ proof reading exonuclease). The enzyme from Pyroccocus furiosus is inhibited by the presence of uracil in template strands. This research has shown that a single uracil located as far as 134 bases ahead of the primertemplate junction causes inhibition of replication. Further, using replication fork mimics, it is shown that, as expected, uracil on a template strand being copied by Pol D causes inhibition. Surprisingly, though, the presence of uracil on a complementary non-copied strand is also inhibitory. A model for uracil recognition by Pol D is proposed. The biochemical properties of the individual, large and small, subunits of the Pol D heterodimer were analysed. Both subunits were found to possess activity when expressed alone although the activity was greatly reduced compared to the Pol D heterodimer. It was not possible to regain the level of activity observed in the Pol D holoenzyme by mixing the two subunits in vitro. This finding contributed to the hypothesis that the carboxyl-terminal region of the large subunit contains an Fe-S cluster that is lost when the protein is purified aerobically. Attempts were made to express Pol D in archaeal hosts and purify the protein with the correct metallo-status; regrettably, these were not successful. Two thermostable bacterial family-B (pol II) DNA polymerases were cloned and expressed in E.coli and their biochemical properties analysed. The enzymes were found to possess many properties that make them amenable to biotechnology: polymerase activity, 3’-5’ proofreading activity, high fidelity rates and the ability to bypass uracil located in template strand DNA. Unfortunately, thermostability assays revealed that the polymerases denatured on exposure to temperatures ~85°C, making them unsuitable in the PCR. Thus, further manipulation is required to determine whether the polymerases have applications in biotechnology

Site-Directed Mutagenesis to Mutate Multiple Residues in a Single Reaction.

Site-directed mutagenesis (SDM) is a technique that allows mutation of specific nucleotide(s) in a codon to study its functional implications in a protein. Commercial kits are available, which require high-performance liquid chromatography purified oligos for this purpose. These kits are expensive, and they are not very efficient, so one has to sequence several clones to get a desired one. We present here a simple method that requires only crude oligos, commercially available high-fidelity enzymes, and the success rate is close to 100%. In addition, up to 6 different mutations can be introduced in one reaction without causing any fortuitous change in the vector backbone. Using this strategy, we have introduced 32 S/T➔A substitutions in the N-terminus head and 13 changes in the C-terminus tail domain of vimentin

Rapid Assembly of Multiple-Exon cDNA Directly from Genomic DNA

Backgrouud. Polymerase chain reaction (PCR) is extensively applied in gene cloning. But due to the existence of introns, low copy number of particular genes and high complexity of the eukaryotic genome, it is usually impossible to amplify and clone a gene as a full-length sequence directly from the genome by ordinary PCR based techniques. Cloning of cDNA instead of genomic DNA involves multiple steps: harvest of tissues that express the gene of interest, RNA isolation, cDNA synthesis (reverse transcription), and PCR amplification. To simplify the cloning procedures and avoid the problems caused by ubiquitously distributed durable RNases, we have developed a novel strategy allowing the cloning of any cDNA or open reading frame (ORF) with wild type sequence in any spliced form from a single genomic DNA preparation. Methodology. Our Genomic DNA Splicing technique contains the following steps: first, all exons of the gene are amplified from a genomic DNA preparation, using software-optimized, highly efficient primers residing in flanking introns. Next, the tissue-specific exon sequences are assembled into one full-length sequence by overlapping PCR with deliberately designed primers located at the splicing sites. Finally, software-optimized outmost primers are exploited for efficient amplification of the assembled full-length products. Conclusions. The Genomic DNA Splicing protocol avoids RNA preparation and reverse transcription steps, and the entire assembly process can be finished within hours, Since genamic DNA is more stable than RNA, it may be a more practical cloning strategy for many genes, especially the ones that are very large and difficult to generate a full length cDNA using oligo-dT primed reverse transcription. With this technique, we successfully doned the full-length wild type coding sequence of human polymeric immunoglobulin receptor, which is 2295 bp in length and composed of 10 exons. © 2007 An et al.published_or_final_versio