A method for cloning mixtures of long,synthetic oligodeoxynucleotides

A method is described for cloning synthetic oligodeoxynucleotides, which can theoretically be of any length. The method requires only a single oligodeoxynucleotide strand and a vector with two unique restriction sites, one of which is for an enzyme that generates 3′ protruding ends. A mixture of unpurified oligonucleotides containing a wild-type genetic regulatory sequence of the Escherichia coli gnd gene and two mutations of it was cloned into a plasmid carrying a gnd-lacZ protein fusion. Individual cloned oligonucleotides were readily identified by direct DNA sequencing of plasmid templates. The method is rapid, efficient, and has application to gene synthesis and site-directed mutagenesis.     

Method for cloning single-stranded oligonucleotides in a plasmid vector

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.     

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis.     

Universal CG cloning of polymerase chain reaction products

Single-insert cloning of DNA fragments without restriction enzymes has traditionally been achieved using TA cloning, with annealing of a polymerase chain reaction (PCR) fragment containing a single overhanging 3′ A to a plasmid vector containing a 3′ T. In this article, we show that the analogous “CG cloning” is faster and far more efficient, using AhdI to generate a C-vector. For an afternoon ligation, CG cloning achieved double the cloning efficiency and more than 4-fold the number of transformants compared with TA cloning. However, blunt-end ligation was markedly more efficient than both. CG cloning could prove to be extremely useful for single-copy high-throughput cloning.     

Cloning of a plasmid region specifying the N transfer system of bacterial conjugation in Escherichia coli

HindIII restriction sites were created artificially by the insertion of the transposon Tn.5 into the IncN plasmid pCU1 near a presumptive end of its conjugal transfer region (tra). This allowed cloning of an entire and continuous 19.4-kb region of this plasmid that specifies the N transfer system. The cloning vector was the nonconjugative plasmid pACYC184. The recombinant plasmid was as efficient in transfer as the parental N plasmid. Other clones and deletions extending into the tra region allowed localization of a 11.2-kb segment of this region that determines sensitivity to the N-specific bacteriophages IKe and PRD1. It could also be concluded that the ability of pCU1 to promote the killing of Klebsiella pneumoniae requires a 2-kb region that is not part of, but adjacent to the tra region.     

Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3′ end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.     

Cloning and characterization of the termination site tI for the gene int transcript in phage lambda

A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed. In one of these vectors a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM). The drug-resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with either various sticky ends or blunt ends. These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites. Insertion mutants are selected by their resistance to kanamycin. When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated. In addition, two new MCSs having single restriction sites have been formed by altering the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter. The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.     

Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification

Reverse genetics has become pivotal in influenza virus research relying on rapid generation of tailored recombinant influenza viruses. They are rescued from transfected plasmids encoding the eight influenza virus gene segments, which have been cloned using restriction endonucleases and DNA ligation. However, suitable restriction cleavage sites often are not available. Here, we describe a cloning method universal for any influenza A virus strain which is independent of restriction sites. It is based on target-primed plasmid amplification in which the insert provides two megaprimers and contains termini homologous to plasmid regions adjacent to the insertion site. For improved efficiency, a cloning vector was designed containing the negative selection marker ccdB flanked by the highly conserved influenza A virus gene termini. Using this method, we generated complete sets of functional gene segments from seven influenza A strains and three haemagglutinin genes from different serotypes amounting to 59 cloned influenza genes. These results demonstrate that this approach allows rapid and reliable cloning of any segment from any influenza A strain without any information about restriction sites. In case the PCR amplicon ends are homologous to the plasmid annealing sites only, this method is suitable for cloning of any insert with conserved termini.     

RF cloning: a restriction-free method for inserting target genes into plasmids

Restriction-free (RF) cloning provides a simple, universal method to precisely insert a DNA fragment into any desired location within a circular plasmid, independent of restriction sites, ligation, or alterations in either the vector or the gene of interest. The technique uses a PCR fragment encoding a gene of interest as a pair of primers in a linear amplification reaction around a circular plasmid. In contrast to QuickChange site-directed mutagenesis, which introduces single mutations or small insertions/deletions, RF cloning inserts complete genes without the introduction of unwanted extra residues. The absence of any alterations to the protein as well as the simplicity of both the primer design and the procedure itself makes it suitable for high-throughput expression and ideal for structural genomics.     

A physical map of plasmid pDU1 from the cyanobacterium Nostoc PCC 7524

Nostoc 7524 contains three different plasmids of molecular weight, 4, 8, and 28 Mdal. The smallest plasmid, designated pDU1, because of its size and ease of isolation, may prove to be useful as a cloning vector. Plasmid pDU1 was incubated separately with 26 different restriction enzymes and only 8 of the enzymes tested cut pDU1. A composite restriction enzyme map consisting of a total of 17 restriction sites was constructed for BglI, HindIII, HpaI, and XbaI. The sites of restriction enzyme cleavage were determined by single, double, and partial digests of plasmid DNA or redigestion of isolated restriction fragments. All the restriction sites were aligned relative to the single BglI site. This is the first restriction enzyme map of a plasmid from a filamentous cyanobacterium.     

Development of the SapI/AarI Incision Mediated Plasmid Editing Method

Plasmid engineering and molecular cloning is a virtually ubiquitous tool in biology. Although various methods have been developed for ligating DNA molecules or targeted mutagenesis of plasmids, each has its limitations. Many of the commonly used laboratory strategies are inefficient, while commercially available kits are quite costly and often specialized for highly specific circumstances. Here, we describe the SapI/AarI incision mediated plasmid editing (SIMPLE) method, which allows users to perform site-directed mutagenesis, deletions, and even short insertions into any plasmid in a single PCR reaction, using just one restriction enzyme. In addition, the SIMPLE method can be adapted to insert any sized DNA fragment into a vector using a two-step PCR approach, and can be used to ligate any number of DNA fragments with non-compatible ends in the specific order desired. The SIMPLE method provides researches an efficient and powerful tool with a broad range of applications for molecular cloning.     

Efficient procedure for site-directed mutagenesis mediated by PCR insertion of a novel restriction site

Better understanding of proteins'' structure/function relationship and dissecting their functional domains are still challenges yet to be mastered. Site-directed mutagenesis approaches that can alter bases at precise positions on the gene sequence can help to reach this goal. This article describes an efficient strategy that can be applied not only for both deletion and substitution of target amino acids, but also for insertion of point mutations in promoter regions to study cis-regulating elements. This method takes advantage of the plasticity of the genetic code and the use of compatible restriction sites.Key words: site-directed mutagenesis, restriction site, cloning, PCRUnderstanding the proteins structure/function relationship and dissecting their functional domains is one of the biggest challenges to current proteomic studies.¹ This is mainly achieved by site-directed mutagenesis experiments that can alter bases at precise positions on the gene sequence.² Modifying DNA sequences has become feasible with PCR amplification.³ During the last decade, several strategies have been developed to simplify this approach and increase its efficiency.⁴ The introduction of a site-directed mutation can be realized by one or more PCR reactions. Most of the strategies used in site-directed mutagenesis are based on a substitution of a single base, which leads to a change in one amino acid. This article describes an efficient strategy that can be applied for either deletion or substitution of target amino acids. This strategy is based on performing PCR reactions to create a new restriction site in the sequence of origin, corresponding to the desired mutation. The choice of the restriction site to be created depends on the nature of the amino acid that one desires to introduce in the protein sequence. Since such restriction sites may extend beyond the mutated codon. The preservation of the other codon is done by taking advantage of the plasticity of the genetic code where one amino acid can be encoded by multiple codons.This method was performed in two steps (Fig. 1). In the first step, the DNA sequence of interest, cloned in a plasmid, served as a template for two PCR reactions. Two PCR products are generated. The first one consists of the beginning of the sequence, from the start codon to the mutagenized amino acid codon, where the forward primer bears the start codon region and the reverse primer bears the newly introduced restriction site at the same location of the mutagenized codon. The second PCR product consists of the end of the coding sequence, from the mutagenized amino acid codon to the stop codon. This fragment is generated using a forward primer bearing the same new restriction site as the first PCR product''s reverse primer, and a reverse primer bearing the stop codon region. The two PCR products were cloned separately into a vector in the appropriate orientation. In the second step, the cloning vector bearing the first PCR product was digested with a restriction enzyme site in the vector, and by the restriction enzyme corresponding to the restriction site created by the reverse primer used in the PCR reaction. The resulting fragment was cloned into the vector containing the second PCR fragment, predigested with same two restriction enzymes. The whole mutagenized coding sequence is reassembled by in-frame subcloning of the 3′ end of the coding sequence downstream the 5′ end. All the PCR products were generated using the high fidelity Pfu DNA Polymerase (Promega, Madison, WI USA). For any site-directed mutagenesis experiment, this two-step cloning procedure requires the use of appropriate PCR primers that harbor the desired mutation of the target amino acid. These primers are partially overlapping and contain a common or complementary restriction site enabling the in-frame assembly of the whole coding sequence.Open in a separate windowFigure 1Mutagenesis strategy by restriction enzyme site insertion. (A) In the first step, two PCR products were generated using the full length coding sequence as template. The mutation is carried by the two primers b and c, which are flanked by the same or compatible restriction enzyme''s site (white segment). Both PCR products are separately cloned in the cloning vector in the appropriate orientation. In the second step, the whole mutagenized coding sequence is reassembled by in-frame sub cloning of the 3′ end of the coding sequence downstream the 5′ end. (B) Substitution of threonine by arginine as a result of the insertion of a BglII restriction site. DNA sequencing is carried out to make sure that only the desired change is introduced in the coding sequence. (B-1) The sequence of the native cDNA. (B-2) the sequence of the mutagenized cDNA included BglII restriction site sequence.This approach has been used in a recent study to address the structure/function relationship of the STAS domain of the Arabidopsis thaliana Sultr1;2 sulfate transporter.⁵ A good example of this approach is the replacement of the threonine-serine couple at position 587–588 with an arginine-serine couple. The codon for threonine is: TGT, and that for arginine is: TCT. Serine can be encoded by both TCA and AGA codons. The chosen restriction site used for the reassembly of the whole coding sequence is that of the BglII enzyme: TCT AGA. The insertion of this restriction site enables the substitution of the Thr in position 587 with an Arg while preserving the serine residue in position 588. The BglII restriction site is introduced in the reverse primer and the forward primer used to generate the first and second PCR products respectively. The DNA sequence of the reassembled mutagenized cDNA was checked by sequencing. Than it was expressed, under pGAL1O promoter bearing by pYES2 vector, in yeast mutant deficient in sulfate transporter and the mutagenic protein was detected by imunodetection.Bioinformatic study reveals that this method can be applied to checked a large number of substitutions, insertions or deletions and that finding the right restriction site is not a limiting factor (data no shown).In conclusion, this article describes an efficient two-step procedure for site-directed mutagenesis using primers bearing a restriction site, which is absent from the sequence of origin. The primers flanked by sequences introducing the same or compatible restriction sites mediate the incorporation of the mutation at the selection site. The choice of the restriction site depends on the nature of the desired mutation: insertion, substitution or deletion of an amino acid in a particular position. This strategy can be also used to insert point mutations in promoter regions to study cis-regulating elements.     

Physical mapping of plasmid pDB101: A potential vector plasmid for molecular cloning in streptococci

A physical map of the streptococcal macrolides, lincomycin, and streptogramin B (MLS) resistance plasmid pDB101 was constructed using six different restriction endonucleases. Ten recognition sites were found for HindIII, seven for HindII, eight for HaeII, and one each for EcoRI, HpaII, and KpnI. The localization of the restriction cleavage sites was determined by double and triple digestions of the plasmid DNA or sequential digestions of partial cleavage products and isolated restriction fragments, and all sites were aligned with a single EcoRI reference site. Plasmid pDB101 meets all requirements essential for a potential molecular cloning vehicle in streptococci; i.e., single restriction sites, a MLS selection marker, and a multiple plasmid copy number. The vector plasmid described here makes it possible to clone selectively any fragment of DNA cleaved with EcoRI, HpaII, or KpnI, or since the sites are close to each other in map position, any combination of two of these restriction enzymes.     

Identification of a hotspot for transformation of Neisseria meningitidis by shuttle mutagenesis using signature-tagged transposons

Shuttle mutagenesis using signature-tagged transposons was employed to generate a library of individually tagged mutants of the Neisseria meningitidis strain B1940, which belongs to serogroup B. The use of tagged transposons allowed us to monitor for enrichment for single mutants during the process of shuttle mutagenesis, by amplification of the tags and subsequent sequence determination. Enrichment of a single clone occurred during the transformation of the meningococci with transposon-containing plasmid DNA. Sequence determination around the site of transposon insertion revealed that the transposon had mutagenized a previously unknown locus, which was designated hrtA (high rate of transformation). hrtA-mediated transformation was independent of TnMax5 and tag sequences, and it most probably involved recombination events. The hrtA locus is restricted to meningococci and gonococci and is present in few apathogenic neisserial species. Chromosomal mapping of hrtA and six further hrt sites revealed a random distribution of highly transforming DNA fragments on the meningococcal chromosome. In conclusion, our data demonstrate that shuttle mutagenesis of naturally competent bacteria using signature-tagged transposons allows the isolation of chromosomal DNA fragments, which exhibit a high transformation efficiency, and which, therefore, are likely to be involved in horizontal gene transfer.     

Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii.     

Plasmids with temperature-dependent copy number for amplification of cloned genes and their products

Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30°C these miniplasmids are present in 20–50 copies per cell of Escherichia coli, whereas at temperatures above 35°C the plasmids replicate without copy number control during 2–3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plasmid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded β-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles.     

IRDL Cloning: A One-Tube,Zero-Background,Easy-to-Use,Directional Cloning Method Improves Throughput in Recombinant DNA Preparation

Rapid and efficient construction of expression vectors and subsequent transformation are basic recombinant methods for the investigation of gene functionality. Although novel cloning methods have recently been developed, many laboratories worldwide continue to use traditional restriction digestion-ligation methods to construct expression vectors owing to financial constraints and the unavailability of appropriate vectors. We describe an improved restriction digestion-ligation (IRDL) cloning method that combines the advantage of directional cloning from double digestion-ligation with that of a low background observed by using a positive selection marker gene ccdB to facilitate digestion and ligation in a single tube. The IRDL cloning overcomes the time-consuming and laborious limits of traditional methods, thereby providing an easy-to-use, low-cost, and one-step strategy for directional cloning of target DNA fragments into an expression vector. As a proof-of-concept example, we developed two yeast vectors to demonstrate the feasibility and the flexibility of the IRDL cloning method. This method would provide an effective and easy-to-use system for gene cloning and functional genomics studies.     

Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites

A method is described for the efficient insertion of mutagenic oligodeoxynucleotide cassettes which allow saturation of a target amino acid codon with multiple mutations. Restriction sites are introduced by oligonucleotide-directed mutagenesis procedures to flank closely the target codon in the plasmid containing the gene. The restriction sites to be introduced are chosen based on their uniqueness to the plasmid, proximity to the target codon and conservation of the final amino acid coding sequence. The flanking restriction sites in the plasmid are digested with the cognate restriction enzymes, and short synthetic duplex DNA cassettes (10-25 bp) are inserted. The mutagenic cassette is designed to restore fully the wild-type coding sequence, except over the target codon, and to eliminate one or both restriction sites. Elimination of a restriction site facilitates selection of clones containing the mutagenic oligodeoxynucleotide cassette. To make the cassettes, single-stranded oligodeoxynucleotides and their complements are synthesized in separate pools containing different codons over the target. This method has been successfully applied to generate 19 amino acid substitutions at position 222 in the subtilisin protein sequence.