An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids

We describe an improved, universal method for the seamless integration of DNA fragments into plasmids at any desired position. The protocol allows in vitro joining of insert and linearized plasmid at terminal homology regions using the BD In-Fusion cloning system. According to the standard BD In-Fusion protocol, vectors are linearized by restriction enzyme digestion. Linearization of plasmids by polymerase chain reaction (PCR), instead of restriction enzyme digestion, extends the usefulness of the method by rendering it independent of restriction endonuclease recognition sites and by allowing seamless insertion of DNA fragments at any position, without introduction of unwanted nucleotides flanking the site of insertion. The combination of PCR linearization of plasmids and BD In-Fusion technology has shown to be very useful for the insertion of genes into the expression regions of multiple plasmids for the heterologous expression of proteins in Escherichia coli. Hands-on time is minimal and there is no need for preparative gel electrophoresis. The protocol is very simple and only involves PCR and liquid handling steps. The method should therefore theoretically have a good potential for automation.     

Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase

Here, we describe a method that offers a unique way to engineer plasmids with precision but without digestion using restriction enzymes for the insertion of DNA. The method allows the insertion of PCR fragments in between any two nucleotides within a target plasmid. The only requirement is that the amplified fragments must be embedded between DNA sequences homologous to the site in which the integration is planned. This method is an adaptation of the QuikChange Site-Directed Mutagenesis protocol. It is simpler than the existing cloning strategies and is suitable for multiparallel constructions of new plasmids. We have demonstrated its utility by constructing plasmids in which we have successfully integrated PCR fragments up to 1117 bp.     

Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens.

A procedure is presented, that has allowed the rapid assignment of transposon Tn1 and Tn7 insertion sites in the large (130 Md) nopaline Ti-plasmid pTiC58, to specific restriction enzyme fragments. Total bacterial DNA is isolated from Agrobacterium tumefaciens strain C58 mutants that carry a transposon in their Ti-plasmid, and digested with an appropriate restriction endonuclease. The fragments are separated on an agarose gel, denatured and transferred to nitrocellulose filters. These are hybridized against purified wild type pTiC58, or against segments of PTiC58, cloned in E. coli using pBR322 as a vector plasmid. DNA sequences homologous to the probe are detected by autoradiography, thus generating a restriction enzyme pattern of the plasmid from a digest of total bacterial DNA. Mutant fragments can be readily identified by their different position compared to a wild type reference. This protocol eliminates the need to separate the large plasmid from chromosomal DNA for every mutant. In principle, it can be applied to the restriction enzyme analysis of insertion or deletion mutants in any plasmid that has no extensive homology with the chromosome.     

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.     

A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.     

A Simple CELI Endonuclease-Based Protocol for Genotyping both SNPs and InDels

Modern genetic analyses rely on efficient genotyping of single-nucleotide polymorphisms (SNP) or insertion/deletion length polymorphisms (InDel) in genomes. Methods available to genotype these polymorphisms include sequencing, cleaved amplified polymorphic sequence, high-resolution DNA melting, and microarray analyses, which are all rather tedious or expensive to set up for daily use. Here, we report a simplified label-free CELI endonuclease (CELI)-based protocol that enables us to detect both SNPs and InDels for fragment lengths between 500 and 6 kb. PCR-amplified target DNA fragments were annealed, cleaved by CELI, and analyzed either cost-effectively by agarose gel electrophoresis or automatically by capillary electrophoresis. The optimal amplification sizes, potential blind ends, and the maximum pooling capacities were examined for both electrophoresis protocols. We believe that the CELI-based genotyping protocol can be used in the detection of mutations, marker-assisted breeding, map-based cloning, and genome-wide association studies.     

Site-specific chromosomal integration of large synthetic constructs

We have developed an effective, easy-to-use two-step system for the site-directed insertion of large genetic constructs into arbitrary positions in the Escherichia coli chromosome. The system uses λ-Red mediated recombineering accompanied by the introduction of double-strand DNA breaks in the chromosome and a donor plasmid bearing the desired insertion fragment. Our method, in contrast to existing recombineering or phage-derived insertion methods, allows for the insertion of very large fragments into any desired location and in any orientation. We demonstrate this method by inserting a 7-kb fragment consisting of a venus-tagged lac repressor gene along with a target lacZ reporter into six unique sites distributed symmetrically about the chromosome. We also demonstrate the universality and repeatability of the method by separately inserting the lac repressor gene and the lacZ target into the chromosome at separate locations around the chromosome via repeated application of the protocol.     

Dynamics of <Emphasis Type="Italic">Vulmar/VulMITE</Emphasis> group of transposable elements in <Emphasis Type="Italic">Chenopodiaceae</Emphasis> subfamily <Emphasis Type="Italic">Betoideae</Emphasis>

Transposable elements are important factors driving plant genome evolution. Upon their mobilization, novel insertion polymorphisms are being created. We investigated differences in copy number and insertion polymorphism of a group of Mariner-like transposable elements Vulmar and related VulMITE miniature inverted-repeat transposable elements (MITEs) in species representing subfamily Betoideae. Insertion sites of these elements were identified using a modified transposon display protocol, allowing amplification of longer fragments representing regions flanking insertion sites. Subsequently, a subset of TD fragments was converted into insertion site-based polymorphism (ISBP) markers. The investigated group of transposable elements was the most abundant in accessions representing the section Beta, showing intraspecific insertion polymorphisms likely resulting from their recent activity. In contrast, no unique insertions were observed for species of the genus Beta section Corollinae, while a set of section-specific insertions was observed in the genus Patellifolia, however, only two of them were polymorphic between P. procumbens and P. webbiana. We hypothesize that Vulmar and VulMITE elements were inactivated in the section Corollinae, while they remained active in the section Beta and the genus Patellifolia. The ISBP markers generally confirmed the insertion patterns observed with TD markers, including presence of distinct subsets of TE insertions specific to Beta and Patellifolia.     

Genome-wide analysis of mobile genetic element insertion sites

Mobile genetic elements (MGEs) account for a significant fraction of eukaryotic genomes and are implicated in altered gene expression and disease. We present an efficient computational protocol for MGE insertion site analysis. ELAN, the suite of tools described here uses standard techniques to identify different MGEs and their distribution on the genome. One component, DNASCANNER analyses known insertion sites of MGEs for the presence of signals that are based on a combination of local physical and chemical properties. ISF (insertion site finder) is a machine-learning tool that incorporates information derived from DNASCANNER. ISF permits classification of a given DNA sequence as a potential insertion site or not, using a support vector machine. We have studied the genomes of Homo sapiens, Mus musculus, Drosophila melanogaster and Entamoeba histolytica via a protocol whereby DNASCANNER is used to identify a common set of statistically important signals flanking the insertion sites in the various genomes. These are used in ISF for insertion site prediction, and the current accuracy of the tool is over 65%. We find similar signals at gene boundaries and splice sites. Together, these data are suggestive of a common insertion mechanism that operates in a variety of eukaryotes.     

A modified pBR322 vector with improved properties for the cloning, recovery, and sequencing of blunt-ended DNA fragments

The construction of a plasmid vector which facilitates the cloning and recovery of blunt-ended DNA fragments is described. This plasmid, called pHP34, differs from pBR322 by a 10-bp insertion which introduces a unique SmaI site immediately flanked by two EcoRI sites. Blunt-ended DNA fragments cloned in the SmaI site can be recovered by digestion with EcoRI. Small cloned fragments can be chemically sequenced using a strategy which does not require their purification. The use of a plasmid related to pHP34 for in vitro mutagenesis by the insertion of a DNA linker fragment conferring an antibiotic resistance is also discussed.     

Isolation of RNA and DNA fragments using diatomaceous earth

A series of simple and phenol-free silica-based protocols for isolating and cleaning RNA or DNA fragments from different sources were developed. Cytoplasmic RNA isolated from hybridoma cells by this method was used in RT-PCR. DNA fragments obtained using this protocol were suitable for further subcloning, gene transformation and DNA sequencing. © Rapid Science Ltd. 1998     

Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.     

A rapid method for determining the relationship between cDNA clones

We describe a technique for rapidly screening the inserts of plasmids for homology to each other by using DNA fragments isolated in agarose gels to probe Southern blots of DNA prepared by the "miniprep" alkaline lysis method. The procedure includes a technique for labeling DNA fragments in agarose gel slices without further purification. The protocol results in a significant savings in time and expense and a considerable increase in fragment yield over methods involving fragment purification from polyacrylamide or agarose gels.     

Use of pulsed-field-gradient gel electrophoresis to construct a physical map of the Caulobacter crescentus genome

The restriction enzyme DraI cleaves the Caulobacter crescentus genome into at least 35 fragments which have been resolved in agarose gels using pulsed-field-gradient gel electrophoresis (PFGE). When digests were performed using DNA from strains containing Tn5 insertion mutations, altered band migrations were observed. Using PFGE with the appropriate pulse times, size differences as small as 2% could be resolved in large fragments. Using this approach, we have constructed a partial physical map of the genome which correlates well with the C. crescentus genetic map and have shown the size of the genome to be approx. 3800 kb. Using hybridization with cloned genes, we have determined the map locations of five previously unmapped genes. In addition, we have shown that PFGE can be used to rapidly determine the map locations of new insertion mutations or the sizes of deletion mutations.     

DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism.

We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of an arbitrary PCR for rapid identification of Tn917 insertion sites in Staphylococcus epidermidis: characterization of biofilm-negative and nonmucoid mutants

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.     

The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

Insertional mutagenesis is a productive strategy for the exploration of genetic regulation of important biological and pathological processes, such as tumorigenesis. Successful implementation of this strategy depends heavily on an efficient approach to the identification of insertion sites present in the host genome. Here, we have introduced an easy and efficient protocol, called Adenosine-ended Primer Extension Polymerase Chain Reaction (APE-PCR), which represents several advantages, including the Addition technique we previously developed, primer extension approach coupled with biotin-streptavidin based purification, introduction of nano-scale magnetic particles, and digestion of DNA with a combination of enzymes. We have demonstrated that APE-PCR is able to amplify more and larger specific proviral insertion site (PIS)-derived fragments, with a lower non-specific background produced, fewer steps and less DNA samples required, flexibility in choice of restriction enzymes applied, at a lower cost. Replacement of regular magnetic beads with nano-scale ones in the protocol can further increase its power. Moreover, even with small amount of sample DNA, PISs can be recovered and analyzed. Thus, based on the results provided from this study, we believe that APE-PCR represents an efficient method in mapping of PISs and likely, the insertion sites of other types of DNA elements as well.     

一种适用于片段替换/插入突变扫描的克隆方法

功能序列的改造是基因工程的一项基础技术。序列改造往往涉及较大片段的插入或替换,为了获得更优化的改造序列,这种片段插入或替换经常需要以扫描方式进行。传统的酶切连接方式在这种情况下可能难以实现或者费时费力。建立了一种golden gate方法结合混合克隆和快速鉴定的策略（简称Gmix）,利用该策略,成功地将外源基因（Gaussia荧光素酶基因）以4种模式,扫描式插入或替换HBV复制质粒的指定区域。该方法成功率高,操作简便快速且成本较低,适合于其他DNA序列的类似改造工作。     

More than skin and bones: Comparing extraction methods and alternative sources of DNA from avian museum specimens

Next‐generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol–chloroform and silica column) to three different sources of DNA (toe pad, skin punch and bone) from 10 historical avian museum specimens. We show that a modified phenol–chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol–chloroform protocol, probably as a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol–chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols.