Phenotypic Expression of PCR-Generated Random Mutations in a Pseudomonas putida Gene after Its Introduction into an Acinetobacter Chromosome by Natural Transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37°C. Of recombinant clones that failed to express xylE at 37°C, about 10% expressed the gene when grown at 22°C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.     

T载体介导的基于attL核心区LR反应的简化快速Gateway克隆系统

Gateway克隆技术已得到广泛的应用。该技术先通过BP反应将目标片段连到带有完整attL特异识别位点的入门载体,然后与终载体通过LR反应得到表达载体。Gateway克隆方法与传统的酶切连接方法相比有快速简单等优点。但是,BP和LR酶都非常昂贵。本研究首先对3个常用Gateway载体的atts特异位点序列比对发现,attL序列核心交换位点“core attL”的21~22 bp长的碱基是保守和必要的。由此,设计含有core-attL序列的引物,通过PCR克隆得到DNA片段并连入pMD18-T载体,然后进行LR反应,可成功得到目标表达载体,并在保守的位点上正确重组。本研究还对其中一个带有绿色荧光蛋白基因的表达载体转化至烟草,能够正常表达该蛋白质。结果表明,通过将含有attL核心位点基因片段连接到pMD18-T载体上,可以省略BP反应而将目标片段连接到终载体上,节约了反应时间和成本。     

Pellicle associated adherence film above incubation broth surface - an inexpensive adjunct to recognizing Candida krusei in the laboratory

Background

Targeting Induced Local Lesions in Genomes (TILLING) is a high throughput reverse genetics tool which detects mismatches (single point mutations or small indels) in large number of individuals of mutagenized populations. Currently, TILLING is intensively used for genomics assisted molecular breeding of several crop plants for desired traits. Most commonly used platform for mutation detection is Li-COR DNA Analyzer, where PCR amplified products treated with single strand mismatch specific nuclease are resolved on denaturing gels. The molecular size of any cut product can be easily estimated by comparing with IR dye labeled markers of known sizes. Similar fluorescent dye labeled size markers are also used for several genotyping experiments. Currently, commercially available size standards are expensive and are restricted up to only 700 bp which renders estimation of products of sizes greater than 700 bases inaccurate.

Findings

A simple protocol was developed for labeling 5' end of multiple DNA size markers with fluorescent dyes. This method involves cloning a pool of different size markers of DNA in a plasmid vector. PCR amplification of plasmid using IR dye labeled universal primers generates 5' fluorescent labeled products of various sizes. The size of products constituting the ladder can be customized as per the need. The generated size markers can be used without any further purification and were found to be stable up to one year at -20°C.

Conclusions

A simple method was developed for generating fluorescent dye labeled size standards. This method can be customized to generate different size standards as per experimental needs. The protocol described can also be adapted for developing labeled size standards for detection on platforms other than Li-COR i.e. other than infra red range of the spectrum.     

A rapid method for purification of organelles for DNA isolation: self-generated percoll gradients

Self-generated Percoll gradients have been used for rapid purification of crude chloroplasts and mitochondria, obtained by common differential centrifugation techniques. Such purified organelles were used for isolating DNA from safflower (Carthamus tinctorius L.), carrot (Daucus carota L.), various Solanaceae, and numerous somatic hybrids. The method is simple, has the advantage of not requiring DNase, and is particularly well suited when only limited amounts of aseptically grown shoots are available. As judged by restriction enzyme analyses and chloroplast DNA cloning experiments, the DNAs are of sufficient purity for many molecular biological applications without CsCl gradient purification.     

Dissection of the nuclear genome of barley by chromosome flow sorting

Isolation of mitotic chromosomes using flow cytometry is an attractive way to dissect nuclear genomes into their individual chromosomal components or portions of them. This approach is especially useful in plants with complex genomes, where it offers a targeted and hence economical approach to genome analysis and gene cloning. In several plant species, DNA of flow-sorted chromosomes has been used for isolation of molecular markers from specific genome regions, for physical mapping using polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), for integration of genetic and physical maps and for construction of chromosome-specific DNA libraries, including those cloned in bacterial artificial chromosome vectors. Until now, chromosome analysis and sorting using flow cytometry (flow cytogenetics) has found little application in barley (2n = 14, 1C ∼ 5,100 Mbp) because of the impossibility of discriminating and sorting individual chromosomes, except for the smallest chromosome 1H and some translocation chromosomes with DNA content significantly different from the remaining chromosomes. In this work, we demonstrate that wheat–barley ditelosomic addition lines can be used to sort any arm of barley chromosomes 2H–7H. Thus, the barley genome can be dissected into fractions representing only about 6–12% of the total genome. This advance makes the flow cytogenetics an attractive tool, which may greatly facilitate genome analysis and gene cloning in barley.     

Transfer-PCR (TPCR): a highway for DNA cloning and protein engineering

DNA cloning and protein engineering are basic methodologies employed for various applications in all life-science disciplines. Manipulations of DNA however, could be a lengthy process that slows down subsequent experiments. To facilitate both DNA cloning and protein engineering, we present Transfer-PCR (TPCR), a novel approach that integrates in a single tube, PCR amplification of the target DNA from an origin vector and its subsequent integration into the destination vector. TPCR can be applied for incorporation of DNA fragments into any desired position within a circular plasmid without the need for purification of the intermediate PCR product and without the use of any commercial kit. Using several examples, we demonstrate the applicability of the TPCR platform for both DNA cloning and for multiple-site targeted mutagenesis. In both cases, we show that the TPCR reaction is most efficient within a narrow range of primer concentrations. In mutagenesis, TPCR is primarily advantageous for generation of combinatorial libraries of targeted mutants but could be also applied to generation of variants with specific multiple mutations throughout the target gene. Adaptation of the TPCR platform should facilitate, simplify and significantly reduce time and costs for diverse protein structure and functional studies.     

A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.

A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.     

TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (eg. cDNA, genomic DNA and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3′ sequences of the primers match the template perfectly. When two outer mutagenic oligos are incorporated together with the desired mutagenic oligos into the newly synthesised mutant strand, they serve as anchors for PCR primers which have 3′ sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

A method for clone sequence confirmation using a mismatch-specific DNA endonuclease

Site-directed mutagenesis and polymerase chain reaction (PCR)-based cloning are well-established methods carried out routinely in most modern molecular biology laboratories. Application of these methods requires confirmation of the DNA sequence of the target gene by sequencing of DNA purified from multiple colonies, a laborious process. We have developed an alternative approach to screen DNA amplified directly from colony DNA for both desired and undesired mutations. This approach is based on the use of a plant mismatch DNA endonuclease, Surveyor Nuclease, to directly screen clones derived by site-directed mutagenesis. We have also used this approach to identify error-free clones of three genes from celery cDNA produced by PCR and TOPO cloning. Sequence confirmation using Surveyor Nuclease provides a fast and simple approach to obtain desired clones from site-directed mutagenesis and PCR-based cloning methods without the necessity of sequencing DNAs purified from multiple clones.     

pKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli

The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.     

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans

Aims: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR‐based diagnostic tool for P. penetrans. Methods and Results: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria‐specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Conclusions: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. Significance and Impact of the Study: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro‐organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.     

A novel simple and rapid PCR-based site-directed mutagenesis method

Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector. In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5′ end of one of the primers, whereas the other primer starts with the nucleotide at position (−1) of the one to be modified. The PCR is carried out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat α integrin CD11b A-domain and the human CD8β cloned in pPICZαB, pGEX-2T, and CDM8 expression vectors, respectively.     

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.     

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.     

Defined oligonucleotide tag pools and PCR screening in signature-tagged mutagenesis of essential genes from bacteria

We describe a fast and simple method for signature-tagged mutagenesis (STM) using defined oligonucleotides for tag construction into mini-Tn5 and PCR instead of hybridization for rapid screening of bacterial mutants in vivo. A collection of 12 unique 21-mers were synthesized as complementary DNA strands to tag bacterial mutants constructed by insertional mutagenesis using pUTmini-Tn5Km2 plasmids. Tags were tested in a combination of assays by PCR and compared to hybridization for specificity and for large-scale screening. Each defined tag has the same melting temperature, an invariable region to optimize PCRs and a variable region for specific amplification by PCR. A series of "suicide" plasmids carrying mini-Tn5s, each with a specific tag, were transferred into Pseudomonas aeruginosa, giving 12 libraries of mutants; groups of 12 mutants were pooled and arrayed into 96-well microplates, representing approximately one-sixth of the P. aeruginosa 5.9-Mb genome. This simple STM method can be adapted to any bacterial system and used for genome scanning in various growth conditions.     

Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli

PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5′ termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories.     

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.     

Polishing the craft of genetic diversity creation in directed evolution

Genetic diversity creation is a core technology in directed evolution where a high quality mutant library is crucial to its success. Owing to its importance, the technology in genetic diversity creation has seen rapid development over the years and its application has diversified into other fields of scientific research. The advances in molecular cloning and mutagenesis since 2008 were reviewed. Specifically, new cloning techniques were classified based on their principles of complementary overhangs, homologous sequences, overlapping PCR and megaprimers and the advantages, drawbacks and performances of these methods were highlighted. New mutagenesis methods developed for random mutagenesis, focused mutagenesis and DNA recombination were surveyed. The technical requirements of these methods and the mutational spectra were compared and discussed with references to commonly used techniques. The trends of mutant library preparation were summarised. Challenges in genetic diversity creation were discussed with emphases on creating “smart” libraries, controlling the mutagenesis spectrum and specific challenges in each group of mutagenesis methods. An outline of the wider applications of genetic diversity creation includes genome engineering, viral evolution, metagenomics and a study of protein functions. The review ends with an outlook for genetic diversity creation and the prospective developments that can have future impact in this field.     

Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies

Background  

In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction.