Graphical contig analyzer for all sequencing platforms (G4ALL): a new stand-alone tool for finishing and draft generation of bacterial genomes

Genome assembly has always been complicated due to the inherent difficulties of sequencing technologies, as well the computational methods used to process sequences. Although many of the problems for the generation of contigs from reads are well known, especially those involving short reads, the orientation and ordination of contigs in the finishing stages is still very challenging and time consuming, as it requires the manual curation of the contigs to guarantee correct identification them and prevent misassembly. Due to the large numbers of sequences that are produced, especially from the reads produced by next generation sequencers, this process demands considerable manual effort, and there are few software options available to facilitate the process. To address this problem, we have developed the Graphic Contig Analyzer for All Sequencing Platforms (G4ALL): a stand-alone multi-user tool that facilitates the editing of the contigs produced in the assembly process. Besides providing information on the gene products contained in each contig, obtained through a search of the available biological databases, G4ALL produces a scaffold of the genome, based on the overlap of the contigs after curation.

Availability

The software is available at: http://www.genoma.ufpa.br/rramos/softwares/g4all.xhtml     

Sequence-constructive SELEX: A new strategy for screening DNA aptamer binding to Globo H

We proposed to use a novel stepwise sequence-constructive SELEX method to develop DNA aptamers that can recognize Globo H which is a tumor-associated carbohydrate antigen. A combinatorial synthetic library that consisted of DNA molecules with randomized regions of 15-bases was used as the starting library for the first SELEX procedure. The input DNA library for the second round of SELEX consisted of the extension of the 5′ and 3′-ends with 7-bases that were randomized from four selected aptamers. The third round of SELEX was performed following the same procedures as described for the second round of SELEX. The experimental results indicate that the binding affinity of DNA aptamers to Globo H was enhanced when using the sequence-constructive SELEX approach. The selectivity of the DNA aptamers for related disaccharides, mannose derivatives, and Globo H analogs demonstrated the ability of the DNA aptamers to discriminate the presence of various glycans with different structures.     

DNA fingerprinting: a tool for determining genetic distances between strains of poultry

Summary DNA fingerprinting, a technique based on the detection of hypervariable minisatellite regions in DNA restriction fragments, was tested for its applicability to conduct population genetics in poultry. Using MspI digestion and phage M13 DNA as a probe, between 25 and 35 minisatellite-containing DNA fragments were observed per bird. Comparison of the banding pattern of offspring with their parents revealed that the bands were inherited as stable genetic traits. The variability of the DNA fingerprinting pattern was reduced in inbred strains. DNA fingerprints of chickens from five well-defined populations of known genetic relationships were analyzed and indices of genetic distances were computed. They correctly reflected the history of these strains, indicating that DNA fingerprinting may be a powerful tool to characterize genetic relationships between different breeding populations of the same species.     

DNA barcoding is a useful tool for the identification of marine fishes from Japan

In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification.     

Anti-bromodeoxyuridine monoclonal antibody: an alternative tool for the identification of replicated DNA at the electron microscope level

A new method for identifying the replicated DNA at the electron microscope level is described. Cells were first exposed in vitro to 5-bromodeoxyuridine (BUdR) in conjunction with 5-fluorodeoxyuridine (FUdR) and BUdR incorporated into DNA was then detected on Lowicryl-embedded sections by immunogold technique using a monoclonal anti-BUdR antibody. After using this method, chromatin and chromosomes are strongly labelled.     

A system for sex determination from degraded DNA: a useful tool for palaeogenetics and conservation genetics of ursids

In this paper, we characterise three sex-specific genes (ZFX/Y, SRY, AMLX/Y) for all eight extant bear species and propose a new, robust and accurate molecular procedure to identify the sex of bears from non-invasive samples and fossil remains. These materials contain tiny amounts of poorly preserved deoxyribonucleic acid (DNA), leaving Polymerase Chain Reaction (PCR) amplification very prone to contamination and difficult to analyse. By taking into account the ancient DNA requirements, the duplex procedures that we developed are efficient not only on DNA extracted from bear faeces but also on ancient DNA extracted from a brown bear fossil 7,500 years old. Defined specifically for ursids, the procedure for faecal samples (co-amplification of ZFX/Y and SRY markers) appears more accurate than other published procedures, as it prevents cross-amplification of potential ingested prey and contamination (19 non-ursid species tested). This system can be applied to threatened bear populations to improve the reliability of sex-ratio and population-size estimates based on non-invasive samples. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Base composition of DNA from symbiotic dinoflagellates: a tool for phylogenetic classification

DNA of eight endosymbiotic dinoflagellates (zooxanthellae) from seven different host species has been analyzed as to its thermal characteristics and base composition by means of spectrophotometry and high performance liquid chromatography. All algae under investigation contain both methylcytosine and hydroxymethyluracil in addition to the bases typical of nuclear DNA. As a result, melting temperatures are decreased, suggesting lower contents of guanine plus cytosine than actually present. True percentages of guanine plus cytosine plus methylcytosine range from about 43 to 54 mol%. They are unique for the symbionts from different hosts, indicating phylogenetic separation of the taxa comparised within the genus Symbiodinium.Abbreviations dA deoxyadenosine - dC deoxycytidine - dG deoxyguanosine - dT deoxythymidine - m5dC 5-methyldeoxycytidine - hmdU 5-hydroxymethyldeoxyuridine - rC ribocytidine - Br8G bromine-80guanosine - A adenine - C cytosine - G guanine - T thymine - m5C 5-methylcytosine - hmU 5-hydroxymethyluracil - G+C guanine plus cytosine plus 5-methylcytosine - HPLC high performance liquid chromatography - T _m temperature at the midpoint of hyperchromic shift - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide - EDTA ethylenediamine-tetraacetic acid, disodium salt - TRIS tris-(hydroxymethyl)-aminomethane - 1×SSC standard saline citrate (0.15 M NaCl+0.015 M trisodium citrate, pH 7.0)     

CGcgh: a tool for molecular karyotyping using DNA microarray-based comparative genomic hybridization (array-CGH)

Microarray-based comparative genomic hybridization (array-CGH) is a technique by which variations in copy numbers between two genomes can be analyzed using DNA microarrays. Array CGH has been used to survey chromosomal amplifications and deletions in fetal aneuploidies or cancer tissues. Herein we report a user-friendly, MATLAB-based, array CGH analyzing program, Chang Gung comparative genomic hybridization (CGcgh), as a standalone PC version. The analyzed chromosomal data are displayed in a graphic interface, and CGcgh allows users to launch a corresponding G-banding ideogram. The abnormal DNA copy numbers (gains and losses) can be identified automatically using a user defined window size (default value is 50 probes) and sequential student t-tests with sliding windows along with chromosomes. CGcgh has been tested in multiple karyotype-confirmed human samples, including five published cases and trisomies 13, 18, 21 and X from our laboratories, and 18 cases of which microarray data are available publicly. CGcgh can be used to detect the copy number changes in small genomic regions, which are commonly encountered by clinical geneticists. CGcgh works well for the data from cDNA microarray, spotted oligonucleotide microarrays, and Affymetrix Human Mapping Arrays (10K, 100K, 500K Array Sets). The program can be freely downloaded from . Y. S. Lee and A. Chao contributed equally to this work.     

Base composition analysis of human mitochondrial DNA using electrospray ionization mass spectrometry: a novel tool for the identification and differentiation of humans

In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative. Furthermore, with mtDNA, the clustering of SNPs complicates the design of SNP extension primers or hybridization probes. This article describes an automated electrospray ionization mass spectrometry method that can detect a number of clustered SNPs within an amplicon without a priori knowledge of specific SNP positions and can do so quantitatively. With this technique, the base composition of a PCR amplicon, less than 140 nucleotides in length, can be calculated. The difference in base composition between two samples indicates the presence of an SNP. Therefore, no post-PCR analytical construct needs to be developed to assess variation within a fragment. Of the 2754 different mtDNA sequences in the public forensic mtDNA database, nearly 90% could be resolved by the assay. The mass spectrometer is well suited to characterize and quantitate heteroplasmic samples or those containing mixtures. This makes possible the interpretation of mtDNA mixtures (as well as mixtures when assaying other SNPs). This assay can be expanded to assess genetic variation in the coding region of the mtDNA genome and can be automated to facilitate analysis of a large number of samples such as those encountered after a mass disaster.     

Antioxidant actions of plant foods: Use of oxidative DNA damage as a tool for studying antioxidant efficacy

Plant-food-derived antioxidants and active principles such as flavonoids, hydroxycinnamates (ferulic acid, chlorogenic acids, vanillin etc.), β-carotene and other carotenoids, vitamin E, vitamin C, or rosemary, sage, tea and numerous extracts are increasingly proposed as important dietary antioxidant factors. In this endeavor, assays involving oxidative DNA damage for characterizing the potential antioxidant actions are suggested as in vitro screens of antioxidant efficacy. The critical question is the bioavailability of the plant-derived antioxidants.     

Birding and DNA: species for the new millennium

Capsule Based on the 1999 Witherby Memorial Lecture – reviews how developments in molecular and population genetics have led to a reappraisal of species limits in birds.

The taxonomy of birds of the West Palearctic has moved from the comparative stability of the ‘Voous List’ into a period of serious activity, with new data emerging in almost every issue of every evolutionary and avian journal! This activity comes from two directions. Firstly, developments in population genetics, molecular biology, acoustics, behaviour and distributional studies have opened new avenues to measuring differentiation among groups of birds. This, in turn, has led to the recognition that earlier views of what constitutes a ‘species’ are in need of modification (‘improvement’), and the emergence of the ‘lineage concept’ of species. I review some of the species concepts most relevant to avian studies, and attempt to show how and why this change has happened, and its consequences for taxonomy and species limits. Examples are given in the form of ‘case studies’, and include Carrion/Hooded Crows Corvus corone/cornix, Green-winged/Eurasian Teals Anas carolinensis/crecca and Phylloscopus warblers.     

'FloraArray' for screening of specific DNA probes representing the characteristics of a certain microbial community

To investigate uncharacterized microbial communities, a custom DNA microarray named 'FloraArray' was developed for screening specific probes that would represent the characteristics of a microbial community. The array was prepared by spotting 2000 plasmid DNAs from a genomic shotgun library of a sludge sample on a DNA microarray. By comparative hybridization of the array with two different samples of genomic DNA, one from the activated sludge and the other from a nonactivated sludge sample of an anaerobic ammonium oxidation (anammox) bacterial community, specific spots were visualized as a definite fluctuating profile in an MA (differential intensity ratio vs. spot intensity) plot. About 300 spots of the array accounted for the candidate probes to represent anammox reaction of the activated sludge. After sequence analysis of the probes and examination of the results of blastn searches against the reported anammox reference sequence, complete matches were found for 161 probes (58.3%) and >90% matches were found for 242 probes (87.1%). These results demonstrate that 'FloraArray' could be a useful tool for screening specific DNA molecules of unknown microbial communities.     

DNA barcodes provide new evidence of a recent radiation in the genus Sporophila (Aves: Passeriformes)

The capuchinos are a group of birds in the genus Sporophila that has apparently radiated recently, as evidenced by their lack of mitochondrial genetic diversity. We obtained cytochrome c oxidase I (COI) sequences (or DNA barcodes) for the 11 species of the group and various outgroups. We compared the patterns of COI variability of the capuchinos with those of the largest barcode data set from neotropical birds currently available (500 species representing 51% of avian richness in Argentina), and subjected COI sequences to neighbour-joining, maximum parsimony and Bayesian phylogenetic analyses as well as statistical parsimony network analysis. A clade within the capuchinos, the southern capuchinos, showed higher intraspecific and lower interspecific divergence than the remaining Argentine species. As most of the southern capuchinos shared COI haplotypes and pairwise distances within species were in many cases higher than distances between them, the phylogenetic affinities within the group remained unresolved. The observed genetic pattern is consistent with both incomplete lineage sorting and gene flow between species. The southern capuchinos constitute the only large group of species among the neotropical birds barcoded so far that are inseparable when using DNA barcodes, and one of few multispecies avian groups known to lack reciprocal monophyly. Extending the analysis to rapidly evolving nuclear and mitochondrial markers will be crucial to understanding this radiation. Apart from giving insights into the evolution of the capuchinos, this study shows how DNA barcoding can rapidly flag species or groups of species worthy of deeper study.     

DNA labelling of varieties covered by patent protection: a new solution for managing intellectual property rights in the seed industry

Plant breeders’ rights are undergoing dramatic changes due to changes in patent rights in terms of plant variety rights protection. Although differences in the interpretation of »breeder’s exemption«, termed research exemption in the 1991 UPOV, did exist in the past in some countries, allowing breeders to use protected varieties as parents in the creation of new varieties of plants, current developments brought about by patenting conventionally bred varieties with the European Patent Office (such as EP2140023B1) have opened new challenges. Legal restrictions on germplasm availability are therefore imposed on breeders while, at the same time, no practical information on how to distinguish protected from non-protected varieties is given. We propose here a novel approach that would solve this problem by the insertion of short DNA stretches (labels) into protected plant varieties by genetic transformation. This information will then be available to breeders by a simple and standardized procedure. We propose that such a procedure should consist of using a pair of universal primers that will generate a sequence in a PCR reaction, which can be read and translated into ordinary text by a computer application. To demonstrate the feasibility of such approach, we conducted a case study. Using the Agrobacterium tumefaciens transformation protocol, we inserted a stretch of DNA code into Nicotiana benthamiana. We also developed an on-line application that enables coding of any text message into DNA nucleotide code and, on sequencing, decoding it back into text. In the presented case study, a short command line coding the phrase »Hello world« was transformed into a DNA sequence that was inserted in the plant genome. The encoded message was reconstructed from the resulting T1 seedlings with 100 % accuracy. The feasibility and possible other applications of this approach are discussed.     

Assessment on the binding characteristics of dasatinib,a tyrosine kinase inhibitor to calf thymus DNA: insights from multi-spectroscopic methodologies and molecular docking as well as DFT calculation

Abstract

The binding characteristics of calf thymus DNA (ct-DNA) with dasatinib (DSTN), a tyrosine kinase inhibitor was assessed through multi-spectroscopic methodologies and viscosity measurement combined with molecular docking as well as DFT calculation to understand the binding mechanism, affinity of DSTN onto ct-DNA, effect of DSTN on ct-DNA conformation, and among others. The results confirmed DSTN bound onto ct-DNA, leading to forming the DSTN–ct-DNA complex with the binding constant of 4.82?×?10³ M^?1 at 310?K. DSTN preferentially inserted to the minor groove of ct-DNA with rich A-T region, that was the binding mode of DSTN onto ct-DNA was groove binding. The enthalpic change (ΔH⁰) and entropic change (ΔS⁰) during the binding process of DSTN with ct-DNA were 128.9?kJ mol^?1 and 489.2?J mol^?1 K^?1, respectively, confirming clearly that the association of DSTN with ct-DNA was an endothermic process and the dominative driven-force was hydrophobic interaction. Meanwhile, the results also indicated that there was a certain extent of electrostatic force and hydrogen bonding, but they maybe play an auxiliary role. The CD measurement results confirmed the alteration in the helical configuration of ct-DNA but almost no change in the base stacking after binding DSTN. The results revealed that there was the obvious change in the conformation, the dipole moment, and the atomic charge distribution of DSTN in the B-DNA complexes, compared with free DSTN, to satisfy the conformational adaptation. From the obtained fronitier molecular orbitals of DSTN, it can be inferred that the nature of DSTN alters with the change of the environment around DSTN.

Communicated by Ramaswamy H. Sarma