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Molecular techniques that recover unknown sequences next to a known sequence region have been widely applied in various molecular studies, such as chromosome walking, identification of the insertion site of transposon mutagenesis, fusion gene partner, and chromosomal breakpoints, as well as targeted sequencing library preparation. Although various techniques have been introduced for efficiency enhancement, searching for relevant single molecular event present in a large-sized genome remains challenging. Here, the optimized ligation-mediated polymerase chain reaction (PCR) method was developed and successfully identified chromosomal breakpoints far away from the exon of the new exon junction without the need for nested PCR. In addition to recovering unknown sequences next to a known sequence region, the high efficiency of the method could also improve the performance of targeted  next-generation sequencing (NGS). [ABSTRACT FROM AUTHOR]

Based on the flanking sequence of the agarase gene of FG4 strain, the recombinant plasmid and vector were constructed to obtain the engineering strain which could express the agarase efficiently. In this study, the flanking sequence of FG4 agarase gene is obtained by chromosome walking. The cloned gene is recombined with the expression vector pET-22b (+) and transferred into the recipient BL21. After the successful construction of the expressed strain, the expression of agarase is induced by IPTG and the optimal fermentation conditions of the expressed strain are explored. This strain ensures the catalytic activity of agarase and lays the foundation for the agarasebased pharmaceutical industry, daily chemical industry and food industry. The cloning and expression of agarase gene from Microbulbifers sp. FG4 strain of Microbulbifers provides theoretical support for agarase application in different fields. [ABSTRACT FROM AUTHOR]

Bacillus subtilis BSD-2 produces small molecule antibiotics that effectively control grey mould. To isolate the genes responsible for these antimicrobial activities, a B. subtilis BSD-2 mutant library was constructed utilizing the pHV1249 plasmid with a mini-Tn10 transposon. The antimicrobial potential of the generated strains was evaluated in the presence of cucumber grey mould and the mutant sequences were cloned by chromosome walking. Among the generated mutants, four mutants showed enhanced inhibitory effects in the presence of grey mould, and one mutant lost its inhibitory effect. The negative 1–19 mutant strains showed 98% sequence homology of the yodF gene, which encodes a Na+/metabolite permease. Next, we constructed a yodF knockout vector (pB-y-k) and used it to transform B. subtilis. A mutant yodF strain lost its inhibitory effect on grey mould, whereas reversion of the mutation restored the fungistatic activity.

Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method–integrative modeling of genomic regions (IMGR)–to increase the genomic resolution of our traces to 10 kb. [ABSTRACT FROM AUTHOR]

Highlights • Two new aac(6′)-aph(2″) transposon structures were found in Enterococcus faecalis from Beijing, China. • Truncated aac(6′)-aph(2″) transposons were not always related to high plasmid location rate and high transfer efficiency. • Truncated aph(2″)-Ia region was identified upstream of a type III transposon with different rates among transposon groups. • Truncated aph(2″)-Ia region had similar positive rate trend as transposon plasmid location and conjugation-positive rates. ABSTRACT Transfer of aac(6′)-aph(2″) transposons mediating high-level gentamicin resistance (HLGR) in Enterococcus faecalis is a serious problem in the clinic. However, factors affecting the transfer of aac(6′)-aph(2″) have not yet been elucidated. The current study aimed to examine the genetic and molecular basis of HLGR in E. faecalis strains isolated in Beijing (China) and to clarify the relationship between transfer efficiency of aac(6′)-aph(2″) transposons and the transposon structure/location. A total of five transposon structures were identified by PCR mapping of the corresponding transposon regions, including a Tn 5281 -like non-truncated transposon and four truncated transposons. A plasmid location study of aac(6′)-aph(2″) by Southern blot following S1-PFGE and filter mating conjugation experiments demonstrated that plasmid location rates correlated with conjugation-positive rates. Chromosome walking to identify the sequence upstream of a representative type III truncated transposon found a truncated aph(2″)-Ia region, and further PCR analysis of this region among strains from different groups revealed similar a positive rate trend as the transposon plasmid location rate and conjugation-positive rate. In conclusion, aac(6′)-aph(2″) transposons were of different structures in E. faecalis strains from Beijing, with two new transposon structures that have not been reported elsewhere. Presence of the truncated aph(2″)-Ia region upstream of some truncated transposons suggests recombination between aminoglycoside-modifying enzyme genes. Possible links exist among plasmid location, conjugation and the presence of truncated aph(2″)-Ia upstream of the transposon. [ABSTRACT FROM AUTHOR]

Bacillus subtilis BSD-2 produces small molecule antibiotics that effectively control grey mould. To isolate the genes responsible for these antimicrobial activities, a B. subtilis BSD-2 mutant library was constructed utilizing the pHV1249 plasmid with a mini-Tn10 transposon. The antimicrobial potential of the generated strains was evaluated in the presence of cucumber grey mould and the mutant sequences were cloned by chromosome walking. Among the generated mutants, four mutants showed enhanced inhibitory effects in the presence of grey mould, and one mutant lost its inhibitory effect. The negative 1-19 mutant strains showed 98% sequence homology of the yodF gene, which encodes a Na+/metabolite permease. Next, we constructed a yodF knockout vector (pB-y-k) and used it to transform B. subtilis. A mutant yodF strain lost its inhibitory effect on grey mould, whereas reversion of the mutation restored the fungistatic activity. [ABSTRACT FROM AUTHOR]

Cold environmental conditions influence the growth and development of plants, causing crop reduction or even plant death. Under stress conditions, cold-inducible promoters regulate cold-related gene expression as a molecular switch. Recent studies have shown that the chloroplast-expressed GPAT gene plays an important role in determining cold sensitivity. However, the mechanism of the transcriptional regulation of GPAT is ambiguous. The 5'-flanking region of GPAT with length of 1494 bp was successfully obtained by chromosome walking from Lilium pensylvanicum. The cis-elements of GPAT promoters were predicted and analyzed by a plant cisacting regulatory DNA element database. There exist core promoter regions including TATA-box and CAAT-box and transcription regulation regions, which involve some regulatory elements such as I-box, W-box, MYB, MYC, and DREB. Full-length and four 5'-deletion fragments linked with GUS vectors were constructed and transformed into Nicotiana tabacum by Agrobacterium-mediated transformation. Transient transformation and histochemical staining of leaves indicated that the activity of the GPAT promoter was strong and induced by low-temperature stress. The deletion of a -294 bp region suggested that the DRE-motif was functionally an essential element for cold induction, and the deletion of -1494 bp and -1194 bp regions suggested that negative regulation exists in the promoter. Our results show that the GPAT gene promoter is a key regulator under cold stress and we think that this study will have significant impact on lily molecular breeding and improving the resistance of plants. [ABSTRACT FROM AUTHOR]

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In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum. The objective of this work was to sequence these markers, obtain their flanking regions by chromosome walking and perform an in silico mapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen molecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2-4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize. Two loci associated with apomixis locus were tested in methylation-sensitive RFLP experiments using genomic DNA extracted from leaves. Although both target sequences were methylated no methylation polymorphisms associated with the mode of reproduction were detected.

A chromosome walking method was improved in this work. The new method was named as nonspecific primer anchored PCR (NPA-PCR). Nested gene specific primers were designed based on the known region and long random primer using degeneracy oligonucleotides for nonspecific anchoring. Annealing temperatures were varied to control the priming. Target sequences were obtained by PCR with random primer and gene-specific primer. Nonspecific sequence with long random primers at both ends formed stem loop structure due to inverted terminal repeats. The method was employed to isolate a gene with newly-isolated actinomycin producing strain Streptomyces setonii Z-L-22. A 0.77 kb fragment of actinomycin synthetase gene cluster was isolated from the strain. The fragments of 1474bp and 701bp were obtained, respectively, at the up and down streams of known fragment through the this method. NCBI Blast analysis showed that the walking sequence and the known sequence were located conjointly in the same cluster gene. It demonstrated that the result was correct and this technique could be useful and efficient for chromosome walking or isolating the gene.

Late blight, caused by Phytophthora infestans, is one of the most devastating diseases in cultivated potato. Breeding of new potato cultivars with high levels of resistance to P. infestans is considered the most durable strategy for future potato cultivation. In this study, we report the identification of a new late-blight resistance (R) locus from the wild potato species Solanum bulbocastanum. Using several different approaches, a high-resolution genetic map of the new locus was generated, delimiting Rpi-blb3 to a 0.93 cM interval on chromosome 4. One amplification fragment length polymorphism marker was identified that cosegregated in 1,396 progeny plants of an intraspecific mapping population with Rpi-blb3. For comparative genomics purposes, markers linked to Rpi-blb3 were tested in mapping populations used to map the three other late-blight R loci Rpi-abpt, R2, and R2-like also to chromosome 4. Marker order and allelic conservation suggest that Rpi-blb3, Rpiabpt, R2, and R2-like reside in the same R gene cluster on chromosome 4 and likely belong to the same gene family. Our findings provide novel insights in the evolution of R gene clusters conferring late-blight resistance in Solanum spp.

SiteFinding-PCR is a method for isolating flanking sequence tags (FSTs) of T-DNA insertion lines, but the efficiency needs to be improved. Here we report a computation-assisted design for the random primers used in SiteFinding- PCR. A short sequence, GCATG, was screened from the rice genome and used as the 3′ end of the random primer. When applying the optimized primer for isolating FSTs from 168 transgenic rice lines, we obtained 107 specific products, including 64 FSTs. The efficiency of obtaining FSTs using the modified version of SiteFinding-PCR increased by 73.0% compared with the method previously reported (P < 0.01, µ test). We also provide computational results for several other plant species such as maize, sorghum, Arabidopsis, foxtail millet, and Brachypodium based on the available genome data, so that the modified method could be easily adapted to other species.

The exogenous fragment sequence and flanking sequence between the exogenous fragment and recombinant chromosome of transgenic wheat B102-1-2 were successfully acquired using genome walking technology. The newly acquired exogenous fragment encoded the full-length sequence of transformed genes with transformed plasmid and corresponding functional genes including ubi, vector pBANF-bar, vector pUbiGUSPlus, vector HSP, reporter vector pUbiGUSPlus, promoter ubiquitin, and coli DH1. A specific polymerase chain reaction (PCR) identification method for transgenic wheat B102-1-2 was established on the basis of designed primers according to flanking sequence. This established specific PCR strategy was validated by using transgenic wheat, transgenic corn, transgenic soybean, transgenic rice, and non-transgenic wheat. A specifically amplified target band was observed only in transgenic wheat B102-1-2. Therefore, this method is characterized by high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B102-1-2. [ABSTRACT FROM AUTHOR]

Artemisia annua is still the only commercial source of Artemisinin. To date, a number of biochemical and molecular studies about Artemisinin's biosynthetic pathway have been carried out. In metabolic engineering approach, isolation and characterization of promoters leads to an understanding of which cis-acting elements are responsible for the regulation of gene expression. DBR2 is a key enzyme in Artemisinin biosynthetic pathway. In order to allow chromosome walking beyond the 5'-flanking region of DBR2, two specific primers were used in combination with 6 arbitrary primers in TAIL-PCR method. A 696bp upstream of DBR2 start codon was isolated and cloned. The subsequent sequence analysis using bioinformatics softwares revealed that there were several cis-acting elements such as TATA-box, CAAT-box, and MeJA-responsive element, and several W-box and light-responsive elements inside the DBR2 promoter. These results can be helpful in understanding of artemisinin biosynthesis regulation and will facilitate metabolic engineering of the compound. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Based on a partialsubtilisin-like protease, Pr1 genomic sequence of Pythium guiyangense which has been cloned before, Panhandle PCR strategy was used to amplify the upstream flanking sequence adjacent to the known sequence of the Pr1 gene. Methods: The genomic DNA was firstly digested with Bam H I and then treated with calf intestinal alkaline phosphatase(CIAP). Next, a 5′ phosphorylated oligonucleotide was ligated to the 5′ ends of Bam H I -digested DNA. After denaturation, intrastrand annealing and polymerase extension, a pan with a handle was formed, and lastly the nested PCR was performed. Results: A 864 bp product was amplified, which was adjacent to the known sequence of Pr1 gene. The gene has been accessed by GenBank (Accession:JQ975036). Conclusion: Panhandle PCR is a quick and convenient approach for amplifying and identifying unknown partner genes, which facilitates cloning full-length Pr1 gene. [Copyright &y& Elsevier]

Exogenous fragment sequence and flanking sequence between exogenous fragment and recombinant chromosome of transgenic wheat B72-8-11b were successfully acquired through PCR amplification with cross-matched primers from exogenous genes. Newly acquired exogenous fragment covered the full-length sequence of transformed genes such as transformed plasmid and corresponding functional genes including marker uidA, promoter ubiquitin, lacZ, 1Dx5, and part of sequence of the wheat genome. A specific PCR detection method for transgenic wheat B72-8-11b strain was established on the basis of primers designed according to flanking sequence. The designed primers revealed specific amplification of 132 bp product of transgenic wheat B72-8-11b strain. This method is characteristics of high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B72-8-11b strain. [ABSTRACT FROM AUTHOR]

Abstract: A-T linker adapter polymerase chain reaction (PCR) was modified and employed for the isolation of genomic fragments adjacent to a known DNA sequence. The improvements in the method focus on two points. The first is the modification of the PO4 and NH2 groups in the adapter to inhibit the self-ligation of the adapter or the generation of nonspecific products. The second improvement is the use of the capacity of rTaq DNA polymerase to add an adenosine overhang at the 3′ ends of digested DNA to suppress self-ligation in the digested DNA and simultaneously resolve restriction site clone bias. The combination of modifications in the adapter and in the digested DNA leads to T/A-specific ligation, which enhances the flexibility of this method and makes it feasible to use many different restriction enzymes with a single adapter. This novel A-T linker adapter PCR overcomes the inherent limitations of the original ligation-mediated PCR method such as low specificity and a lack of restriction enzyme choice. Moreover, this method also offers higher amplification efficiency, greater flexibility, and easier manipulation compared with other PCR methods for chromosome walking. Experimental results from 143 Arabidopsis mutants illustrate that this method is reliable and efficient in high-throughput experiments. [Copyright &y& Elsevier]

The barley mutant allele sdw3 confers a gibberellin-insensitive, semi-dwarf phenotype with potential for breeding of new semi-dwarfed barley cultivars. Towards map-based cloning, sdw3 was delimited by high-resolution genetic mapping to a 0.04 cM interval in a 'cold spot' of recombination of the proximal region of the short arm of barley chromosome 2H. Extensive synteny between the barley Sdw3 locus ( Hvu_sdw3) and the orthologous regions ( Osa_sdw3, Sbi_sdw3, Bsy_sdw3) of three other grass species ( Oryza sativa, Sorghum bicolor, Brachypodium sylvaticum) allowed for efficient synteny-based marker saturation in the target interval. Comparative sequence analysis revealed colinearity for 23 out of the 38, 35, and 29 genes identified in Brachypodium, rice, and Sorghum, respectively. Markers co-segregating with Hvu_sdw3 were generated from two of these genes. Initial attempts at chromosome walking in barley were performed with seven orthologous gene probes which were delimiting physical distances of 223, 123, and 127 kb in Brachypodium, rice, and Sorghum, respectively. Six non-overlapping small bacterial artificial chromosome (BAC) clone contigs (cumulative length of 670 kb) were obtained, which indicated a considerably larger physical size of Hvu_sdw3. Low-pass sequencing of selected BAC clones from these barley contigs exhibited a substantially lower gene frequency per physical distance and the presence of additional non-colinear genes. Four candidate genes for sdw3 were identified within barley BAC sequences that either co-segregated with the gene sdw3 or were located adjacent to these co-segregating genes. Identification of genic sequences in the sdw3 context provides tools for marker-assisted selection. Eventual identification of the actual gene will contribute new information for a basic understanding of the mechanisms underlying growth regulation in barley. [ABSTRACT FROM AUTHOR]

The article provides the results of the cloning and sequence analysis of the cyanovirin-N homology (CVNH) genomic DNA in Ceratopteris thalictroides. Total genomic DNA was extracted from the leaves of Ceratopteris thalictroides and was investigated using spectrophotometer and polymerase chain reaction, among which. After data analysis, it was revealed that increases at N- and C-terminal regions do occur during the evolution of plant CVNHs.

Abstract: The thermostable direct hemolysin coded by the tdh gene is a marker of virulent strains of Vibrio parahaemolyticus. The tdh genes are flanked by insertion sequences collectively named as ISVs or their remnants; but the ISVs so far examined have accumulated mutations in the transposase genes and underwent structural arrangements and their transposition activity could not be expected; the tdh gene was thus considered to have been acquired by V. parahaemolyticus through horizontal transfer in the past during evolution. We recently isolated from the same patient tdh + strains and a tdh − strain (PCR examination) that were otherwise indistinguishable. The purpose of this study was to examine the hypothesis that the tdh − strain was derived from the tdh + strain by a deletion of the tdh gene mediated by a functional ISV. Southern blot hybridization showed tdh + sequences in the tdh − strain (PSU-1466). Nucleotide sequence analysis of the tdh and its flanking sequences revealed the tdh gene was split into two parts and they were located 3182-bp apart in PSU-1466. The two tdh sequences were flanked by one of the ISVs, named as ISVpa3, in PSU-1466. This genetic structure could be explained by an ISVpa3-mediated partial tdh deletion from a tdh + strain followed by transposition of the duplicated ISVpa3 and the deleted tdh sequence into a neighboring location. The ISVpa3 of PSU-1466 coded for a full-length transposase and a DDE motif. We were able to demonstrate transposition activity of the ISVpa3 cloned from PSU-1466 using the replicon fusion assay with the conjugal transfer of a cointegrate from Escherichia coli to V. parahaemolyticus. Our data support ISVpa3-mediated partial tdh deletion resulted in the emergence of the tdh − strain. [Copyright &y& Elsevier]

In this paper, the basic principle of chromosome walking is presented and we used an actin gene of radiata pine ( Pinus radiata) as an example to conduct upstream and downstream chromosome walking for EST sequences. The full genomic sequence (2154 bp) of the actin gene, including promoters 5′ UTR, CDS and 3′ UTR, was identified by chromosome walking. PCR amplification and DNA band sequencing from 200 unrelated radiata pine trees revealed a total of 21 SNPs for the actin gene, three in the promoter region, 15 in CDS and 4 in 3′ UTR. The results of this experiment provide a technical framework for SNPs discovery in none coding regions of candidate genes. [ABSTRACT FROM AUTHOR]

Abstract: PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio. [Copyright &y& Elsevier]

Abstract: The increased demand for enzymes with new properties makes indispensable the development of easy and rapid strategies to obtain complete genes of new enzymes. Here a strategy is described which includes screening by PCR of new subtilases mediated by Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP) and an improved genome walking method to obtain the complete sequence of the identified genes. Existing methods of genome walking have many limitations, which make them inefficient and time consuming. We have developed an improved genome walking method with novel advances to get a simple, rapid and more efficient procedure based on cassette-ligation. Improvements consist basically in the possibility of a genomic DNA digestion with any restriction enzyme, blunting and 3′ adenylation of digested DNA by Taq DNA polymerase to avoid self-circularization, followed by TA ligation of the adenine 3′ overhanging end to the same unphosphorylated oligo-cassette. The efficiency of the genome walking method was demonstrated by finding the unknown ends of all gene fragments tested, previously obtained by CODEHOP-mediated PCR, including three subtilases (P4, P6 and P7), one xylanase and one lipase, from different strains of Antarctic marine bacteria. [Copyright &y& Elsevier]

One of the most intriguing phenomena of fleshy fruit is the ability to maintain high water content at maturity, even following harvest. This is accomplished by a fruit cuticle that is highly impermeable to water diffusion. In this paper, we report on a novel genotype of tomato, developed via introgression from the wild species Solanum habrochaites, which is characterized by microfissuring of the fruit cuticle and dehydration of the mature fruit. The microfissure/dehydration phenotype is inherited as a single gene, termed Cwp1 (cuticular water permeability). The gene was fine mapped, and its identity was determined by map-based cloning and differential expression analysis in near-isogenic lines. Causality of the Cwp1 gene was shown by the heterologous transgenic expression of the gene in the cultivated tomato, which caused a microfissured fruit cuticle leading to dehydrated fruit. Cwp1 encodes for a protein of unidentified function in the DUF833 domain family. The gene is expressed in the fruit epidermis of the dehydrating genotype harbouring the wild-species introgression, but not in the cultivated tomato. It is expressed only in the primitive green-fruited wild tomato species, but is not expressed in the cultivated Solanum lycopersicum and the closely related Solanum cheesmaniae and Solanum pimpinellifolium, indicating a pre-adaptive role for Cwp1 silencing in the evolution and domestication of the cultivated tomato. [ABSTRACT FROM AUTHOR]

The nucleotide sequence was established for the operon of the Sse9I type II restriction-modification system of Sporosarcina species 9D. The enzymes of the Sse9I system recognize the 5′-AATT-3′ tetranucleotide. The operon includes three genes, sse9IC-sse9IR-sse9IM, which are transcribed unidirectionally and code, respectively, for the controller protein (C. Sse9I), restriction endonuclease (R. Sse9I), and DNA methyltransferase (M. Sse9I). The region immediately upstream of sse9IC was found to contain a conserved nucleotide sequence (C box) providing a binding site for C. Sse9I. The amino acid sequences of C. Sse9I and R. Sse9I were compared with those of related proteins. In the case of R. Sse9I, the highest homology was observed with the R. MunI (5′-CAATTG-3′) and R. EcoRI (5′-GAATTC-3′) regions that harbor the amino acid residues involved in recognizing the AATT inner tetranucleotide. The sse9IR gene was cloned in an expression vector, and recombinant R. Sse 9I was isolated. [ABSTRACT FROM AUTHOR]

Abstract: Ligation-mediated suppression PCR (LMS-PCR) is a powerful tool for walking in unknown genomic DNA regions from known adjacent sequences. This approach has made it feasible to obtain promoter sequences and to enable researchers to identify full-length gene sequences or isoforms of multigene families. However, the advantages of LMS-PCR can be obviated by the presence of incomplete base modifications on the suppression adapters. We propose here that a ‘partial-complementary adapter’ is a more reliable suppression adapter, demanding only 5′-end phosphorylation. We also describe a simplified procedure for the easier preparation of PCR templates with very small quantities of DNA and a fast and direct characterization of the suppression-PCR products. A set of practical guidelines is proposed for pre-checking the efficiency of the adapter modification using two model systems: bacteriophage λ (λ) and Arabidopsis. [Copyright &y& Elsevier]