A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis

A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli β-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification.     

Simplified gene synthesis: a one-step approach to PCR-based gene construction

PCR-based gene synthesis conventionally requires two steps: first, all overlapping oligonucleotides are assembled by self-priming; then an additional pair of primers is used to amplify the full-length gene product. Here we propose a simplified method of gene synthesis which combines these two steps into one. We have found that the efficiency of this one-step method, which we term "Simplified Gene Synthesis", is affected by multiple parameters of the PCR reactions. In particular, the choice of polymerase is critical for successful one-step assembly. Other important factors include the concentration of assembly oligonucleotides and amplification primers. Moreover, we offer a general method to estimate, given a known mutation rate, how many clones should be sequenced in order to be confident of obtaining at least one correct gene product. Having determined the accuracy of gene products synthesized under optimal conditions with Simplified Gene Synthesis, we show that our estimation works well. Overall, the simplified gene synthesis provides an easier and more efficient approach to gene synthesis, providing a further step towards the future goal of generalized automation for this process.     

Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene codon modification

Gene synthesis technologies provide a powerful tool for increasing protein expression through codon optimization and gene modification. Here we describe an improved PCR-based gene synthesis technology, which is accurate, simple and cheap. The improved PCR-based gene synthesis (IPS) method consists of two steps. The first one is the synthesis of 300-400 bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15 bp overlap. The second one is assembling of fragments from the first step into the full-length gene by PCR reaction. Using this approach, we have successfully synthesized a modified phytase gene with 1256 bp in length with optimal codons for expression in Pichia pastoris. P. pastoris strain that expressed the modified phytase gene (phyA-mod) showed a 50% increase in phytase activity level. In addition, we propose an inexpensive method for error correction, based on overlap-extension PCR (OE-PCR).     

A rapid and efficient method for multiple-site mutagenesis with a modified overlap extension PCR

A rapid and efficient method to perform site-directed mutagenesis based on an improved version of overlap extension by polymerase chain reaction (OE-PCR) is demonstrated in this paper. For this method, which we name modified (M)OE-PCR, there are five steps: (1) synthesis of individual DNA fragments of interest (with average 20-bp overlap between adjacent fragments) by PCR with high-fidelity pfu DNA polymerase, (2) double-mixing (every two adjacent fragments are mixed to implement OE-PCR without primers), (3) pre-extension (the teams above are mixed to obtain full-length reassembled DNA by OE-PCR without primers), (4) synthesis of the entire DNA of interest by PCR with outermost primers and template DNA from step 3, (5) post-extension (ten cycles of PCR at 72°C for annealing and extension are implemented). The method is rapid, simple and error-free. It provides an efficient choice, especially for multiple-site mutagenesis of DNAs; and it can theoretically be applied to the modification of any DNA fragment. Using the MOE-PCR method, we have successfully obtained a modified sam1 gene with eight rare codons optimized simultaneously. Electronic Supplementary Material Supplementary material is available for this article at .     

A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are ~500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5–7 days) and suitable for synthesizing long segments of DNA (5–6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.     

Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.     

人端粒酶RNA基因的克隆与鉴定

以人血基因组ＤＮＡ为模板,合成两段２０个寡聚核苷酸为引物,经过ＰＣＲ扩增,得到４８０ｂｐ的片段,克隆到ｐＭＤ１８－Ｔ载体中,经电泳、酶切、ＰＣＲ鉴定后测定序列。序列分析表明氙克隆的人端粒酶ＲＮＡ（ｈｕｍａｎ ｔｅｌｏｍｅａｓｅ ＲＮＡ,ｈＴＲ）基因含有４８０ｂｐ,包括约４５０ｂｐ的编码模板区主序列和约３０ｂｐ的上游调控区序列,其中模板区的１１个核苷酸（５’－ＣＵＡＡＣＣＣＵＡＡＣ－３’）合成端粒亚     

Specificity of the Pseudomonas aeruginosa PAO1 lipoprotein I gene as a DNA probe and PCR target region within the Pseudomonadaceae.

The lipoprotein I gene (oprI) of Pseudomonas aeruginosa PAO1 was cloned and sequenced. A high degree of homology was found between our cloned PAO1 gene sequence and two published oprI sequences. Specific oligonucleotides were designed to amplify the oprI gene by the polymerase chain reaction (PCR). The potential of either the complete gene sequence or the specific oligonucleotide primers as a tool for rapid strain identification was directly assessed against bacterial colonies by PCR or against purified genomic DNA by Southern blot analysis, using a number of representative strains within the Pseudomonadaceae. The oprI gene was found to be well conserved within RNA group I.     

Detection of Bonamia ostreae based on small subunit ribosomal probe

Bonamia ostreae is a protozoan parasite of the flat oyster, Ostrea edulis, which has caused significant loss of oysters in Europe over the last decade. B. ostreae was purified from infected flat oysters and DNA was extracted. The nearly complete small subunit rDNA gene of B. ostreae was amplified using universal oligonucleotides and the PCR product was cloned and sequenced. BLAST research with this sequence revealed similarities to Haplosporidium nelsoni, Haplosporidium costale, and Minchinia teredinis. These data suggest that B. ostreae may be included in the genus Haplosporidium. Specific B. ostreae primers were designed for labeling, by PCR, a probe. This probe was successfully used by in situ hybridization to detect B. ostreae in infected fiat oysters, thus confirming the accuracy of this SSU rDNA sequence. The probe lead also to the detection of Bonamia sp. in infected Tiostrea chilensis and H. nelsoni in infected Crassostrea virginica but not Mikrocytos mackini infected Crassostrea gigas. These primers were also used to detect B. ostreae from infected oyster tissues by PCR. This B. ostreae SSU rDNA gene sequence provides genetic information as a first step toward elucidation of the taxonomic boundaries among the microcell organisms. Moreover, the development of DNA detection assays will be valuable specific diagnostic tools.     

Synthesis and evaluation of phosphorescent oligonucleotide probes for hybridisation assays

Monofunctional, p-isothiocyanatophenyl-derivatives of platinum (II)-coproporphyrin-I (PtCP-NCS) were evaluated as phosphorescent labelling reagents for synthetic oligonucleotides containing a 3′- or 5′-amino modification. Synthesis and purification conditions were optimised to generate high yields and purity of PtCP-labelled oligonucleotide probes. Phosphorescent properties of the PtCP label have been shown to be largely unaffected by conjugation to oligonucleotides of various length, GC composition and label attachment site. 5′-PtCP-labelled oligonucleotides were shown to work efficiently as primers in a standard PCR. A dedicated 532 nm laser-based time-resolved fluorescence plate reader enabled highly sensitive detection of PtCP-labelled oligonucleotides and PCR products, both in solution and in agarose gels, with limits of detection in the order of 0.3 pM. A model system employing two complementary oligonucleotides labelled with PtCP and QSY® 7 dye (dark quencher) showed strong (~20-fold) and specific proximity quenching of PtCP label upon hybridisation in solution. The potential applications of PtCP-labelled probes in hybridisation assays were discussed.     

Polymer supported synthesis of aminooxyalkylated oligonucleotides,and some applications in the fabrication of microarrays

A new protocol has been described for solid phase preparation of 3′- and 5′-aminooxylalkylated oligonucleotides using commercially available reagents. This involves attachment of linker 4 either with an LCAA-CPG support via succinoylation followed by synthesis (3′-aminooxyalkylated oligomers) or formation of its phosphoramidite 6 followed by coupling with desired oligomer (for generating 5′-aminooxyalkylated oligomers). Both the routes produced modified oligonucleotides in sufficiently high yields and purity (on HPLC) via conventional oligonucleotide synthesis on an automated synthesizer and deprotection step using aqueous ammonia (16 h, 60 °C). Aminooxyalkylated oligonucleotides were used to construct microarrays on glass surface (biochips). The performance of the biochips was evaluated by immobilizing modified oligonucleotides on epoxylated glass microslides under different sets of conditions with respect to pH, temperature and time. Further, the constructed microarrays were successfully used for detection of nucleotide mismatches and bacterial typhoid.     

基孔肯雅病毒非结构蛋白基因合成中多位点缺失突变的校正方法

目的:建立一种PCR方法,以快速校正基孔肯雅病毒非结构蛋白基因合成过程中发生的多位点缺失突变。方法:用PCR方法合成基孔肯雅病毒非结构蛋白基因;对测序的克隆进行序列比对,分析不同克隆上缺失突变发生的位置,以保守区域互相重叠的寡核苷酸为上下游引物、以该区域测序正确的克隆为模板进行PCR扩增,得到所需片段,再将这些片段用PCR方法进一步组装成完整的基因序列并进行测序。结果:测序结果表明,经过2次PCR扩增,校正了基孔肯雅病毒非结构蛋白基因合成过程中发生的5个位点缺失突变。结论:得到序列正确的基孔肯雅病毒非结构蛋白基因。在进行基因合成过程中如发生多位点缺失突变,可利用该方法同时对以上突变进行校正,无须再合成引物,降低了实验操作难度,并提高了实验效率。     

Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.     

Insertional gene synthesis, a novel method of assembling consecutive DNA sequences within specific sites in plasmids. Construction of the HIV-1 tat gene.

The construction of the HIV-1 tat gene using a novel method termed insertional gene synthesis (IGS) is described. IGS is used to assemble a gene or any DNA sequence in a stepwise manner within a plasmid containing a single stranded DNA phage origin of replication. The IGS method is based upon consecutive targeted insertions of long DNA oligonucleotides (greater than 100 bases) within the plasmid by oligonucleotide-directed mutagenesis. IGS therefore involves synthesis of only a few oligonucleotides corresponding to one strand of a gene. Furthermore, the gene is synthesized directly adjacent to bacterial gene regulatory sequences for direct expression. Using this approach, the 261 bp tat gene was assembled in three successive cycles adjacent to the lac promoter in the pEMBL-derivative, pKH125. The 15 kD tat protein was produced from this synthetic gene in E. coli upon IPTG induction. However, it was necessary to tightly control the expression of tat by including the lac I gene directly within the tat expression vector.     

Oligonucleotide microarrays: immobilization of phosphorylated oligonucleotides on epoxylated surface

A facile and efficient method for direct immobilization of phosphorylated oligonucleotides on an epoxy-activated glass surface is described. The new immobilization strategy has been analyzed for its performance in DNA microarray under both microwave and thermal conditions. It reflects high immobilization efficiency ( approximately 23%), and signal-to-noise ratio ( approximately 98) and resulted in high hybridization efficiency ( approximately 36%) in comparison to those obtained with standard methods, viz., NTMTA ( approximately 9.76%) and epoxide-amine ( approximately 9.82%). The probes immobilized through the new strategy were found to be heat-stable, since the performance of microarray decreased by only approximately 7% after subjecting it to 20 PCR-like heat cycles, suggesting that the chemistry could be used in integrated PCR/microarray devices. The immobilization of probes following the proposed chemistry resulted in spots of superior quality in terms of spot morphology, spot homogeneity, and signal reproducibility. The constructed microarrays have been successfully used for the discrimination of nucleotide mismatches. In conclusion, these features make the new immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.     

A new method for the synthesis of a structural gene.

A novel method of synthesizing a structural gene or gene fragment, consisting of the first synthesis of a single-stranded DNA (ssDNA), has been developed. As a preliminary test of this method, four synthetic genes or gene fragments have been synthesized. The first one with 396 base pairs (b.p.) codes for the mature rbcS from wheat, the next two with 370 and 342 b.p. respectively, for two half molecules of a gene for trichosanthin and the last one with 315 b.p. for the N-terminal 1-102 residues of human prourokinase. In all these syntheses, a plus-stranded DNA of the target gene was generally assembled by a stepwise or one step T4 DNA ligase reaction of six oligonucleotides (A, *pB, *pC, *pD, *pE and *pF) of 30-71 nucleotides long in the presence of two terminal complementary oligonucleotides (Ab' and eF') and three short inter-fragment complementary oligonucleotides (bc, cd and de). After purification, the synthetic ssDNA was inserted into a cloning vector, pWR13. The resulting product was directly used to transform a host cell. The structure of the cloned synthetic gene was confirmed by DNA sequence analysis.     

PCR fragmentation of DNA

A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

Detection of Streptococcus pneumoniae DNA by using polymerase chain reaction and microwell hybridization with Europium-labelled probes

The present paper describes a novel modification of polymerase chain reaction (PCR) for the detection of Streptococcus pneumoniae DNA in clinical specimens. PCR was based on the detection of a 209-base pair segment of the S. pneumoniae pneumolysin gene. For the demonstration of the amplification product, microwell hybridization with a Europium-labelled oligonucleotide probe complementary to a biotinylated strand of the PCR product was performed, and the presence of the PCR product was monitored by time-resolved fluorescence (TRF) of the Europium chelate. The sensitivity of the assay for purified S. pneumoniae DNA was 50 fg DNA corresponding to 20 genome equivalents of S. pneumoniae DNA. The efficiency of the hybridization step was monitored by using known amounts of synthetic target oligonucleotides as standards. Sensitivity of 3×10⁸ molecules per individual reaction well was achieved with a 30-min attachment time and a 3-h hybridization time.

Detection of PCR-amplified products by the microwell hybridization technique and TRF was compared to agarose gel electrophoresis in 50 middle ear fluid samples obtained from children with acute otitis media. The agarose gel and TRF detection methods identified all culture-positive samples, but both were also positive for 55% of the culture-negative samples. The results suggest that the detection of amplified PCR products by microwell hybridization using Europium-labelled oligonucleotides is a reliable method for the demonstration of the pneumolysin gene fragment. Furthermore, the method is suitable for automation and, thus, for testing high numbers of samples. The clinical significance of the PCR findings remains to be studied.     

Quikgene: a gene synthesis method integrated with ligation-free cloning

Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides or intermediate fragments. Here we present an efficient method that integrates gene synthesis and cloning into one step. Our method, which is evolved from QuikChange mutagenesis, can modify, extend, or even de novo synthesize relatively large genes. The genes are inserted directly into vectors without ligations or subcloning. We de novo synthesized a 600-bp gene through multiple steps of polymerase chain reaction (PCR) directly into a bacterial expression vector. This outside-in gene synthesis method is called Quikgene. Furthermore, we have defined an overlap region of a minimum of nine nucleotides in insertion primers that is sufficient enough to circularize PCR products for efficient transformation, allowing one to significantly reduce the lengths of primers. Taken together, our protocol greatly extends the current length limit for QuikChange insertion. More importantly, it combines gene synthesis and cloning into one step. It has potential applications for high-throughput structural genomics.