快捷的定点突变效率近100%的大引物PCR方法

报道了一种新的PCR突变方法,它不需要纯化大引物或设计特别的旁侧引物．利用一个诱变引物和两个测序引物(T_m≤58℃)作为旁侧引物．第一轮PCR产物12.5 μl直接加入到50 μl的第二轮PCR反应体系作为模板和大引物,在开始第二轮PCR反应时,增加在68℃退火温度下进行10个循环的不对称PCR,这一步骤大大提高了通过600 bp或800 bp大引物所导致的突变效率．结果表明,该方法的产物能够达到高保真、97%～98%的突变效率和高产率．     

在单个PCR管内快捷完成定点突变

采用大引物方法,利用质粒多克隆位点两侧的普通测序引物作为旁侧引物,在单个PCR管内,经2个步骤共34个循环进行定点突变. 该方法通过优化模板和引物的量达到降低PCR循环次数, 通过加入10个在68℃复性条件下的PCR循环达到增加突变效率而无需胶纯化.本方法达到平均62%的突变效率,而且全长扩增产物的产率很高.     

A novel PCR strategy for high-efficiency, automated site-directed mutagenesis

We have developed a novel three-primer, one-step PCR-based method for site-directed mutagenesis. This method takes advantage of the fact that template plasmid DNA cannot be efficiently denatured at its reannealing temperature (T_ra), which is otherwise a troublesome problem in regular PCR. Two flanking primers and one mutagenic primer with different melting temperatures (T_m) are used together in a single PCR tube continuously without any intervention. A single-stranded mutagenic DNA (smDNA) is synthesized utilizing the high T_m mutagenic primer at a high annealing temperature, which prevents the priming of the low T_m primers (i.e. the two flanking primers). A megaprimer is then produced using this smDNA as the template at a denaturing temperature that prevents wild-type template DNA activity. The desired mutant DNA is then obtained by cycling again through these first two steps, resulting in a mutagenic efficiency of 100% in all tested cases. This highly automated method not only eliminates the necessity of any intermediate manipulation and accomplishes the mutagenesis process in a single round of PCR but, most notably, enables complete success of mutagenesis. This novel method is also both cost and time efficient and fully automated.     

Construction of tandem repeats of DNA fragments by a polymerase chain reaction-based method

We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71?bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.     

Rapid and Efficient Gene Splicing Using Megaprimer-Based Protocol

Megaprimer-based methodology has been widely applied in site-directed mutagenesis, but rarely used in gene splicing. In this article, we describe a modification of the megaprimer PCR method, which can efficiently create and amplify a specific ligated chimeric gene segment in a PCR reaction and under a common PCR program that is widely used by researchers. More importantly, this modified method for splicing two or more gene fragments together revealed the mechanism of the megaprimer PCR method, by elucidating the key factor in the megaprimer-based protocol. In this method, the denatured megaprimer divided into two strands. One strand was used as template DNA to regenerate megaprimer and the other strand was used as an oligonucleotide primer to create a ligated chimeric gene product. In this article, we detail the modified megaprimer protocol for creating and amplifying these chimeric gene products, including a specific protocol for large chimeric gene products. We also provide additional tips to increase specificity and efficiency of the protocols. In conclusion, the improved megaprimer PCR protocol is a simple, broadly applicable protocol for splicing two different gene fragments together without relying on restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

邻近多位点基因突变的组合大引物PCR策略(英文)

报道了一种新的组合多位点突变策略,通过单管中三阶段聚合酶链式反应(PCR)得以实现。在第一阶段,PCR扩增出多位点突变大引物,然后在第二阶段延伸大引物,在第三阶段获得全长突变基因序列。基于退火温度与热循环参数的组合大引物反应的优化,三个阶段中退火温度差异小(低于10°C),成功扩增出多位点突变基因序列和比邻突变序列。是一种简单、高效的多位点突变方法。     

Megaprimer-based methodology for deletion of a large fragment within a repetitive polypyrimidine-rich DNA

Site-directed mutagenesis is often a prerequisite for elucidation of the functional significance of cis- and trans-factors involved in gene regulation. The aim of this study was to delete the primary binding site for heterogeneous nuclear ribonucleoprotein I (hnRNPI) within the inducible nitric oxide synthase (iNOS) 3′ untranslated region mRNA. The binding site consists of a 53-nucleotide CU-rich region within a long stretch of polypyrimidines. As a result of primer pair annealing, the repetitive sequence limited the use of several deletion methods based on polymerase chain reaction. Therefore, a megaprimer approach was chosen. The megaprimer was produced by a forward primer outside the polypyrimidine-rich region, and a mutagenic reverse primer annealing to flanking regions of the desired deletion, thereby looping out the target sequence. Subsequently, this megaprimer was used to create the final deletion recombinant. The deletion was verified by sequencing and by ultraviolet cross-linking mouse liver protein extracts with radiolabeled mutant and wild-type RNAs. In conclusion, the megaprimer method offers a solution for generating large internal deletions in repetitive sequences, which facilitates investigations on large repetitive DNA or RNA regions interacting with trans-factors.     

The "megaprimer" method of site-directed mutagenesis

We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.     

An efficient one-step site-directed and site-saturation mutagenesis protocol

We have developed a new primer design method based on the QuickChange™ site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange™ protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries.     

巨型引物PCR法对抗CD3改型单链抗体

亲和力是影响改型单链抗体应用于临床的重要因素之一.利用巨型引物PCR定点诱变方法,设计并化学合成出两组含多个突变位点的简并引物,在第一轮PCR中使用简并引物分别扩增出含突变碱基的两条特异性的DNA片段,即巨型引物,将其经琼脂糖凝胶电泳分离纯化后,作为3′和5′的两端引物应用于第二轮PCR反应中.通过改变标准PCR反应条件,调整引物与模板的浓度,扩增出特异性较强的目的DNA条带.PCR产物经回收后,进行DNA测序.测序结果表明利用该方法扩增得到特异的抗CD3改型单链抗体的突变体库.     

A high-throughput genome-walking method and its use for cloning unknown flanking sequences

We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5′ ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5′ end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5′ flanking regions/promoters of selected plant genes.     

Start Codon Targeted (SCoT) Polymorphism: A Simple,Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants

Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions. A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes. This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose gel electrophoresis. This method was validated in rice using a genetically diverse set of genotypes and a backcross population. Reproducibility was evaluated by using duplicate samples and conducting PCR on different days. Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility. SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers. We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis.     

Long Distance Multiple-Site Directed Plasmid Mutagenesis by One-Step PCR Using Non-overlapped Primers

Site-directed mutagenesis is a very important technique in molecular biological researches. We have developed a new method for long distance multiple-site plasmid mutation by one-step PCR using non-overlap primers. These primers were carefully designed and contained desired mutations in the middle of the primers flanked with 18–25 bp of correct sequence. One pair of the primers was able to generate a short megaprimer. Decreases in the concentrations of these primers increased efficiency of the multiple-site plasmid mutation. All of the mutant PCRs were performed at a common annealing temperature at 55 °C. This method could be widely used in all multiple-site plasmid mutations.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method. Joaquin Sanchis, Layla Fernández, and J. Daniel Carballeira contributed equally.     

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function.     

Single-step overlap-primer-walk polymerase chain reaction for multiple mutagenesis without overlap extension

We present a simple, single-step, single-tube, and rapid method for introducing a series of mutations into cloned DNA. Polymerase chain reaction (PCR)-based mutagenesis methods have become very prevalent due to their simplicity and efficiency for introducing mutations. Our method, overlap-primer-walk PCR, has several advantages over other published methods. It uses two common oligodeoxyribonucleotides and a series of overlapping primers specific for various mutations. Once common flanking primers are selected, two to three mutations require only one additional primer. Therefore, this method is very useful for introduction of multiple mutations in various sites of the target DNA. We illustrate the usefulness of the method by introducing several mutations into the human TNF-α encoding gene.     

Open reading frame analysis by selective PCR-mediated deletion mutagenesis

Recently, we demonstrated that a nested set of DNA fragments can be obtained by using one specific primer and one semirandom primer in a polymerase chain reaction (PCR). We now describe a strategy for selective deletion mutagenesis that is based on this observation. The gene of interest is cloned as a fusion construct with a selectable marker in a small vector, allowing for PCR amplification of the entire recombinant plasmid. The specific primer is complementary to the vector sequence beyond the gene of interest and is oriented downstream. The 3′ end of the semirandom primer is complementary to a triplet (GAT) that is scattered over the entire open reading frame (ORF). It is shown by nucleotide sequence analysis that deletion mutants result exclusively from annealing of the semirandom primer at different GAT triplets. PCR products resulting fro from annealing to GAT triplets elsewhere in the plasmid are counterselected by the need for replication functions and for the expression of the selectable marker. This technique is demonstrated on the Saccharomyces cerevisiae ORF YCL56C.     

Molecular analysis of the accumulation of the transcripts of the large subunit gene of ribulose-1,5-bisphosphate carboxylase/oxygenase by light

Twenty primers of 20 mer referred to universal rice primer (URP) were developed from a repetitive sequence of rice genome. URP-PCR protocol employed stringent PCR with high annealing temperature throughout the thermo-cycling reaction, giving high reproducibility. Under the PCR condition, each single URP primer produced characteristic fingerprints from diverse genomes containing 14 plants, 7 animals and 6 microbes, indicating its universal applicability. The generality of URP-PCR was demonstrated by applying it to 15 cultivars from five rice species, 23 isolates in four Alternaria species producing host-specific toxins on different host plants and 12 bacterial strains including Escherichia coli, Salmonella spp., and Blucella abortus. PCR approach using URP primers will be useful for studying DNA diversity of most eukaryotic or prokaryotic genomes, especially at inter- and intraspecies levels.     

A multiplex PCR test for determination of mating type applied to the plant pathogens Tapesia yallundae and Tapesia acuformis

A multiplex PCR test for determining mating type of the pathogens Tapesia yallundae and Tapesia acuformis is described. The test involves three primers: a "common" primer annealing to DNA sequence conserved in the flanking region of both mating-type idiomorphs and two specific primers annealing to sequence in either the MAT-1 or the MAT-2 idiomorphs. Locating the specific primers in different positions relative to the common primer yielded PCR products of 812 or 418 bp from MAT-1 and MAT-2 isolates, respectively. The test was used successfully to determine the mating type of 118 isolates of T. yallundae and T. acuformis from Europe, North America, and New Zealand. Isolates of both mating types were found on all continents for both species despite the rarely observed occurrence of sexual reproduction of T. acuformis. The multiplex test design should be applicable to other ascomycete species, of use in studies of MAT distribution and facilitating sexual crossing by identifying compatible isolates.