Supported PCR: an efficient procedure to amplify sequences flanking a known DNA segment

We describe a novel modification of the polymerase chain reaction for efficient in vitro amplification of genomic DNA sequences flanking short stretches of known sequence. The technique utilizes a target enrichment step, based on the selective isolation of biotinylated fragments from the bulk of genomic DNA on streptavidin-containing support. Subsequently, following ligation with a second universal linker primer, the selected fragments can be amplified to amounts suitable for further molecular studies. The procedure has been applied to recover T-DNA flanking sequences in transgenic tomato plants which could subsequently be used to assign the positions of T-DNA to the molecular map of tomato. The method called supported PCR (sPCR) is a simple and efficient alternative to techniques used in the isolation of specific sequences flanking a known DNA segment.     

Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences

Transposases mediate transposition first by binding specific DNA end sequences that define a transposable element and then by organizing protein and DNA into a highly structured and stable nucleoprotein 'synaptic' complex. Synaptic complex assembly is a central checkpoint in many transposition mechanisms. The Tn5 synaptic complex contains two Tn5 transposase subunits and two Tn5 transposon end sequences, exhibits extensive protein-end sequence DNA contacts and is the node of a DNA loop. Using single-molecule and bulk biochemical approaches, we found that Tn5 transposase assembles a stable nucleoprotein complex in the absence of Tn5 transposon end sequences. Surprisingly, this end sequence-independent complex has structural similarities to the synaptic complex. This complex is the node of a DNA loop; transposase dimerization and DNA specificity mutants affect its assembly; and it likely has the same number of proteins and DNA molecules as the synaptic complex. Furthermore, our results indicate that Tn5 transposase preferentially binds and loops a subset of non-Tn5 end sequences. Assembly of end sequence-independent nucleoprotein complexes likely plays a role in the in vivo downregulation of transposition and the cis-transposition bias of many bacterial transposases.     

Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR

A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.     

Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained.     

Identification of DNA sequences flanking T-DNA insertions by PCR-walking

In recent years, concerns over genetic modification issues have resulted in regulatory authorities requiring comprehensive analysis of transgene insertion events in the plants that are to be commercialized. Determining that plants are devoid of vector backbone sequences is a trivial task that is best achieved by Southern blot analysis; however, identifying the DNA sequences flanking the T-DNA insertions can be arduous. In this paper, we present a robust method of characterizing this insertion event. We have applied and modified a genomic walking method that combines vectorette and suppression PCR walking.     

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.     

An inverse PCR technique to rapidly isolate the flanking DNA of Dictyostelium insertion mutants

Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum. In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site. For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA. This is a time-consuming process that requires Southern blot analysis of the mutant DNA. In addition, plasmid rescue requires transformation into highly competent Escherichia coli. Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination. We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.     

利用Inverse PCR快速筛选单个T—DNA拷贝转基因水稻植株的方法

In order to obtain single T-DNA copy transgenic rice, we have established a quick method to estimate the T-DNA copy number in transgenic rice using inverse PCR (IPCR). IPCR was used to amplify junction fragments, i.e. plant genomic DNA sequences flanking the known T-DNA sequences, which will help to estimate the T-DNA copy number in transgenic rice. We have analyzed 20 transgenic plants of 15 transgenic lines. Most plants (12) contain one integrated T-DNA copy per genome, 3 plants contain two and 1 plant contains 3 copies. In 4 transgenic plants no T-DNA copies could be detected using this method. The IPCR results were further tested by Southern analysis and sequence analysis.     

Coupling sequences flanking Tn916 do not determine the affinity of binding of integrase to the transposon ends and adjacent bacterial DNA

Coupling sequences are the 6 bp flanking the conjugative transposon Tn916 and are thought to play a role in determining the frequency of conjugative transposition. The affinity of binding of a chimeric protein, which consisted of maltose binding protein fused to the carboxy-terminal DNA binding domain of Tn916 integrase (Int), to different double-stranded oligonucleotide substrates containing coupling sequences associated with high- and low-frequency conjugative transposition was measured using a competition binding assay. The relative affinity of the chimeric protein was unaffected by the nature of the coupling sequences tested. The same results were obtained when the coupling sequences were placed in a different surrounding sequence context. It therefore appears that the effects of different coupling sequences on the frequency of conjugative transposition are not due simply to differences in Int binding.     

Chased PCR: A modified inverse PCR technique to characterize flanking regions of AT-rich DNA fragments

The chased polymerase chain reaction (PCR) technique described here is a convenient method enabling the characterization of flanking regions of a known A/T-rich DNA fragment in two different successive steps. The first step includes a modified inverse PCR with inverted tail-to-tail primers, each with 35 overhanging nucleotides for the insertion into a carrier plasmid. The second step consists of a technique similar to a site-directed mutagenesis. The chased PCR technique is simple, quick, versatile and efficient; it improves the inverse PCR technique and may be applied to any ligation-linker method. Consequently, the techniques for PCR-based gene isolation are more suitable for the isolation of missing sequences of A/T-rich or complex DNA.     

Construction of a deletion library using a mixture of 5'-truncated primers for inverse PCR (IPCR).

A quick in vitro mutagenesis method for the construction of nested deletion libraries was developed. Many deletions can be obtained in a single inverse PCR (IPCR) by replacing one of the two primers with a mixture of 5'-truncated oligodeoxynucleotides. Since chemical DNA synthesis proceeds from the 3'to the 5'end, such a mixture of 5'-truncated oligodeoxynucleotides can easily be obtained in a single automated DNA synthesis under reduced coupling efficiency. This deletion mutagenesis method yields many different deletions in a defined short DNA segment and is, therefore, best suited for a deletion analysis at base pair level. Applications might include functional analysis of regulatory DNA segments and protein engineering work that requires libraries for the expression of N-terminal, C-terminal or internal truncated proteins as well as fusion proteins having different splice sites.     

Streptococcus faecalis R plasmid pJH1 contains an erythromycin resistance transposon (Tn3871) similar to transposon Tn917

The R plasmid pJH1 contains a 5.1-kilobase transposon ( Tn3871 ) that mediates inducible resistance to erythromycin. Three AvaI digestion fragments from this transposon are identical in size to and homologous with three AvaI-derived fragments from the previously described erythromycin resistance transposon Tn917 . These three DNA fragments account for greater than 90% of both transposons.     

Construction of a physical map of a kanamycin (Km) transposon, Tn5, and a comparison to another km transposon, Tn903

Summary A cleavage map of Tn5, a kanamycin (Km) transposon from plasmid JR67, was constructed from pMKI, a composite plasmid of ColE1 and Tn5, and compared to that of Tn903, a Km transposon from plasmid R6-5. The two transposons showed marked heterogeneity in both the structural gene for Km resistance and the inverted repeat regions as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution for the two Km transposons.     

DNA sequences at the sites of three insertions of the transposable element Tn5 in the histidine operon of Salmonella

Summary The DNA sequence has been determined at the sites of three independent occurrences of the transposable element Tn5 in the hisG gene of the histidine operon. All three insertion sites are adjacent to G+C-rich stretches of DNA. In all three cases sequences at the sites of insertion show homology with a region near the ends of Tn5.     

优化的反向PCR结合TAIL-PCR法克隆棉花线粒体atpA双拷贝基因及其侧翼序列

双拷贝基因及其侧翼序列的克隆是分子生物学中的一个难点。将优化的反向PCR(Inverse PCR,iPCR)与TAIL-PCR相结合,有效地克隆双拷贝基因及其侧翼序列。先用Southern blotting方法确定一种能获得合适长度片段的限制性内切酶,然后用优化的iPCR方法对该酶切产物进行自连和扩增,将2个拷贝的侧翼序列区分开。根据iPCR结果,进一步用TAIL-PCR扩增更远侧翼的序列。利用这套方法,获得了棉花可育胞质和不育胞质线粒体双拷贝atpA基因的所有EcoR I限制片段(2.2～5.1 kb)和HindⅢ限制片段(8.5～11.7 kb),克隆到2个拷贝各自的侧翼序列。研究结果说明,优化的iPCR与TAIL-PCR相结合是克隆双拷贝基因及其侧翼序列的一种高效方法。     

A-T linker adapter polymerase chain reaction for determining flanking sequences by rescuing inverse PCR or thermal asymmetric interlaced PCR products

The polymerase chain reaction (PCR)-based genome walking method has been extensively used to isolate unknown flanking sequences, whereas nonspecific products are always inevitable. To resolve these problems, we developed a new strategy to isolate the unknown flanking sequences by combining A-T linker adapter PCR with inverse PCR (I–PCR) or thermal asymmetric interlaced PCR (TAIL–PCR). The result showed that this method can be efficiently achieved with the flanking sequence from the Arabidopsis mutant and papain gene. Our study provides researchers with an additional method for determining genomic DNA flanking sequences to identify the target band from bulk of bands and to eliminate the cloning step for sequencing.     

Multiple homoplasious insertions and deletions of a Triticeae (Poaceae) DNA transposon: a phylogenetic perspective

Background  

Stowaway elements are short, non-autonomous DNA transposons categorized as miniature inverted-repeat transposable elements (MITEs). The high MITE copy number in grass genomes suggests an active history of amplification and insertion, but ongoing MITE activity has only rarely been seen, and ongoing Stowaway activity has never been observed. Thus, a phylogenetic perspective on presence vs. absence of elements in an aligned data set can provide valuable historical insights into the dynamics of MITE acquisition and loss.