Similarity analysis for DNA sequences based on chaos game representation. Case study: The albumin

Using chaos game representation we introduce a novel and straightforward method for identifying similarities/dissimilarities between DNA sequences of the same type, from different organisms. A matrix is associated to each CGR pattern and the similarities result from the comparison between the matrices of the sequences of interest. Three different methods of analysis of the resulting difference matrix are considered: a 3-dimensional representation giving both local and global information, a numerical characterization by defining an n-letter word similarity measure and a statistical evaluation. The method is illustrated by implementation to the study of albumin nucleotides sequences from eight mammal species taking as reference the human albumin.     

Singular value decomposition of 3-D DNA melting curves reveals complexity in the melting process

The thermal denaturation of synthetic deoxypolynucleotides of defined sequence was studied by a three dimensional melting technique in which complete UV absorbance spectra were recorded as a function of temperature. The results of such an experiment defined a surface bounded by absorbance, wavelength, and temperature. A matrix of the experimental data was built, and analyzed by the method of singular value decomposition (SVD). SVD provides a rigorous, model-free analytical tool for evaluating the number of significant spectral species required to account for the changes in UV absorbance accompany-ing the duplex – to – single strand transition. For all of the polynucleotides studied (Poly dA – Poly dT; [Poly (dAdT)]₂; Poly dG – Poly dC; [Poly(dGdC)]₂), SVD indicated the existence of at least 4 – 5 significant spectral species. The DNA melting transition for even these simple repeating sequences cannot, therefore, be a simple two-state process. The basis spectra obtained by SVD analysis were found to be unique for each polynucleotide studied. Differential scanning calorimetry was used to obtain model free estimates for the enthalpy of melting for the polynucleotides studied, with results in good agreement with previously published values. Received: 16 April 1997 / Accepted: 9 July 1997     

DNA序列分析用于常见致病真菌鉴定和分型

随着真菌感染的增多,仅用表型方法鉴定环境中或临床上的致病真菌不足以快速准确地诊断真菌感染疾病,近年来,分子生物学方法因快速、准确而逐步得到应用,其中DNA序列分析已成为鉴定致病真菌到种水平的重要方法。现就DNA序列分析在常见致病真菌分类鉴定及基因分型的应用加以综述。     

Statistical analysis of DNA sequences in the neighborhood of splice sites

Prediction of gene sequences and their exon-intron structure in large eukaryotic genomic sequences is one of the central problems of mathematical biology. Solving this problem involves, in particular, high-accuracy splice site recognition. Using statistical analysis of a splice site-containing human gene fragment database, some characteristic features were described for nucleotide sequences in the splicing site neighborhood, the frequencies of all nucleotides and dinucleotides were determined, and those with frequencies increased or decreased in comparison to a random sequence were identified. The results can be used in sequence annotation, splicing site prediction, and the recognition of the gene exon-intron structure.     

Monitoring of fluorescence during DNA melting as a method for discrimination and detection of PCR products in variety identification

DNA melting curves of genotype-specific PCR fragments were used to differentiate between species and amongst varieties of cereals. Melting curves were generated by ramping the temperature of PCR fragments through their dissociation temperature in the presence of a double-stranded DNA binding dye. Genotypes were discriminated by differences in the position and shape of the melting curve which is a function of the fragment's sequence, length and GC content. Amplification of 5S ribosomal RNA genes generated species-specific fragments for six of the major cereal crops. Of the 15 possible pairwise comparisons, 13 distinctions could be reliably made using melting curve position data. Wheat varieties were identified by the melting profiles of PCR products generated using microsatellite primers. DNA melting curve analysis was conveniently coupled with capillary-PCR using a LightCycler instrument to provide a rapid method of genotyping in cereals.     

Use of mathematically enhanced spectral analysis and spectral contrast techniques for the liquid chromatographic and capillary electrophoretic detection and identification of pharmaceutical compounds

The use of mathematically enhanced ultraviolet/visible (UV/VIS) absorbance spectral analysis and spectral contrast software techniques in high performance liquid chromatography (HPLC) and micellar electrokinetic capillary electrophoresis (MECC) as an aid for the determination of peak homogeneity, identification, and tracking during method development was investigated. Various structurally similar pharmaceutical compounds, and compounds present as either cis/trans isomers, diastereomers, or enantiomers were used as test compounds to probe the limits of this technique. Two tricyclic antidepressants, nortriptyline and imipramine, were employed to study the effects of HPLC mobile phase composition and pH on the ability to identify and track peaks during method development. It was found that method changes altered the spectral matches used for identification, but not enough to cause incorrect peak identification. It was also shown using HPLC that the cis/trans isomers of doxepin and the diastereomers ephedrine and pseudoephedrine could be distinguished. The mathematically enhanced spectral analysis and spectral contrast software techniques were also employed with MECC. Peaks tracking during method development as pH and the concentration of surfactant changes is shown for a separation of various penicillin type antibiotics. It was shown that during chiral MECC (CMECC) analyses ephedrine/pseudoephedrine diastereomers as well as ephedrine enantiomers could be distinguished. The determination of enantiomers is possible in CMECC since enantiomers are eluted as diastereomeric complexes, as opposed to HPLC where they are eluted in their native state. © 1996 Wiley-Liss, Inc.     

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.     

蛋白质序列的六维表示与相似性分析

给出了蛋白质序列的一种六维表示方法,根据这种表示方法有3种不同表示形式,利用这3种形式来构造距离矩阵的信息熵,然后通过信息熵向量的欧式距离、夹角来比较序列之间的相似性。     

Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer

The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation. To evaluate the influence of potentially destructive processes on plasmid DNA associated with long-term storage, capillary gel electrophoresis (CGE) analysis of the LacZ-expressing pCMVbeta plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at -80 degrees C prevent an increase in open circular (oc) plasmid, preserving the covalently closed circular (ccc) form, which is sought for efficient gene transfer. By contrast, long-term storage of plasmid DNA at 4 degrees C leads to the rapid decline of the ccc form and the increase of oc and linear DNA molecules. The use of naked DNA stored for 1, 2, or 13 months at -80 degrees C showed similar in vivo transfer efficiencies by jet-injection. Therefore, analysis of plasmids by CGE allows the reliable determination of integrity and distribution of the topology of the DNA by quantitative means.     

New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnL approach

The development of DNA barcoding (species identification using a standardized DNA sequence), and the availability of recent DNA sequencing techniques offer new possibilities in diet analysis. DNA fragments shorter than 100-150 bp remain in a much higher proportion in degraded DNA samples and can be recovered from faeces. As a consequence, by using universal primers that amplify a very short but informative DNA fragment, it is possible to reliably identify the plant taxon that has been eaten. According to our experience and using this identification system, about 50% of the taxa can be identified to species using the trnL approach, that is, using the P6 loop of the chloroplast trnL (UAA) intron. We demonstrated that this new method is fast, simple to implement, and very robust. It can be applied for diet analyses of a wide range of phytophagous species at large scales. We also demonstrated that our approach is efficient for mammals, birds, insects and molluscs. This method opens new perspectives in ecology, not only by allowing large-scale studies on diet, but also by enhancing studies on resource partitioning among competing species, and describing food webs in ecosystems.     

A note on the incidence of reverse complementary fungal ITS sequences in the public sequence databases and a software tool for their detection and reorientation

Reverse complementary DNA sequences––sequences that are inadvertently cast backward and in which all purines and pyrimidines are transposed––are not uncommon in sequence databases, where they may introduce noise into sequence-based research. We show that about 1% of the public fungal ITS sequences, the most commonly sequenced genetic marker in mycology, are reverse complementary, and we introduce an open source software solution to automate their detection and reorientation. The MacOSX/Linux/UNIX software operates on public or private datasets of any size, although some 50 base pairs of the 5.8S gene of the ITS region are needed for the analysis.     

Comparative analysis of Borrelia isolates from southeastern USA based on randomly amplified polymorphic DNA fingerprint and 16S ribosomal gene sequence analyses

Fifty-three southern USA Borrelia isolates were characterized using randomly amplified polymorphic DNA fingerprinting analysis (RAPD). Twenty-nine types were recognized among 37 B. andersonii strains, seven types among eight B. bissettii strains, and seven types among seven B. burgdorferi sensu stricto strains. Strain TXW-1 formed a separate RAPD type. Nearly complete sequences of the rrs genes from 17 representative southern Borrelia were determined. The similarity values were found to be 96-100% within the B. burgdorferi sensu lato (s.l.) complex, 94-99% among the relapsing fever borreliae, and 93-99% between the two complexes. Phylogenetic analysis indicated that all the Borrelia strains we analyzed could be divided into two parts: the B. burgdorferi s.l. complex and the relapsing fever borreliae complex. TXW-1 segregated with the North American relapsing fever borreliae and formed a separate subbranch.     

A novel approach to the discovery of survival biomarkers in glioblastoma using a joint analysis of DNA methylation and gene expression

Glioblastoma multiforme (GBM) is the most aggressive of all brain tumors, with a median survival of less than 1.5 years. Recently, epigenetic alterations were found to play key roles in both glioma genesis and clinical outcome, demonstrating the need to integrate genetic and epigenetic data in predictive models. To enhance current models through discovery of novel predictive biomarkers, we employed a genome-wide, agnostic strategy to specifically capture both methylation-directed changes in gene expression and alternative associations of DNA methylation with disease survival in glioma. Human GBM-associated DNA methylation, gene expression, IDH1 mutation status, and survival data were obtained from The Cancer Genome Atlas. DNA methylation loci and expression probes were paired by gene, and their subsequent association with survival was determined by applying an accelerated failure time model to previously published alternative and expression-based association equations. Significant associations were seen in 27 unique methylation/expression pairs with expression-based, alternative, and combinatorial associations observed (10, 13, and 4 pairs, respectively). The majority of the predictive DNA methylation loci were located within CpG islands, and all but three of the locus pairs were negatively correlated with survival. This finding suggests that for most loci, methylation/expression pairs are inversely related, consistent with methylation-associated gene regulatory action. Our results indicate that changes in DNA methylation are associated with altered survival outcome through both coordinated changes in gene expression and alternative mechanisms. Furthermore, our approach offers an alternative method of biomarker discovery using a priori gene pairing and precise targeting to identify novel sites for locus-specific therapeutic intervention.     

A novel approach to the discovery of survival biomarkers in glioblastoma using a joint analysis of DNA methylation and gene expression

Glioblastoma multiforme (GBM) is the most aggressive of all brain tumors, with a median survival of less than 1.5 years. Recently, epigenetic alterations were found to play key roles in both glioma genesis and clinical outcome, demonstrating the need to integrate genetic and epigenetic data in predictive models. To enhance current models through discovery of novel predictive biomarkers, we employed a genome-wide, agnostic strategy to specifically capture both methylation-directed changes in gene expression and alternative associations of DNA methylation with disease survival in glioma. Human GBM-associated DNA methylation, gene expression, IDH1 mutation status, and survival data were obtained from The Cancer Genome Atlas. DNA methylation loci and expression probes were paired by gene, and their subsequent association with survival was determined by applying an accelerated failure time model to previously published alternative and expression-based association equations. Significant associations were seen in 27 unique methylation/expression pairs with expression-based, alternative, and combinatorial associations observed (10, 13, and 4 pairs, respectively). The majority of the predictive DNA methylation loci were located within CpG islands, and all but three of the locus pairs were negatively correlated with survival. This finding suggests that for most loci, methylation/expression pairs are inversely related, consistent with methylation-associated gene regulatory action. Our results indicate that changes in DNA methylation are associated with altered survival outcome through both coordinated changes in gene expression and alternative mechanisms. Furthermore, our approach offers an alternative method of biomarker discovery using a priori gene pairing and precise targeting to identify novel sites for locus-specific therapeutic intervention.     

A phylogenetic analysis of the genus Catellatospora based on 16S ribosomal DNA sequences, including transfer of Catellatospora matsumotoense to the genus Micromonospora as Micromonospora matsumotoense comb. nov.

Phylogenetic studies based on the 16S ribosomal gene sequences showed that members of the genus Catellatospora revealed phylogenetic heterogeneity within the family Micromonosporaceae as well as a heterogeneous menaquinone composition. Among them, Catellatospora matsumotoense was closely related to members of the genus Micromonospora, indicating that this organism should be excluded from the genus Catellatospora. On the basis of classical taxonomic characteristics and phylogenetic evidence, Catellatospora matsumotoense is proposed to be transferred to the genus Micromonospora as M. matsumotoense comb. nov.     

Potential use of DNA barcoding for the identification of Salvia based on cpDNA and nrDNA sequences

An effective DNA marker for authenticating the genus Salvia was screened using seven DNA regions (rbcL, matK, trnL–F, and psbA–trnH from the chloroplast genome, and ITS, ITS1, and ITS2 from the nuclear genome) and three combinations (rbcL + matK, psbA–trnH + ITS1, and trnL–F + ITS1). The present study collected 232 sequences from 27 Salvia species through DNA sequencing and 77 sequences within the same taxa from the GenBank. The discriminatory capabilities of these regions were evaluated in terms of PCR amplification success, intraspecific and interspecific divergence, DNA barcoding gaps, and identification efficiency via a tree-based method. ITS1 was superior to the other marker for discriminating between species, with an accuracy of 81.48%. The three combinations did not increase species discrimination. Finally, we found that ITS1 is a powerful barcode for identifying Salvia species, especially Salvia miltiorrhiza.     

Phylogenetic analysis of Stylosanthes (Fabaceae) based on the internal transcribed spacer region (ITS) of nuclear ribosomal DNA

 Phylogenetic relationships in Stylosanthes are inferred by DNA sequence analysis of the ITS region (ITS1–5.8S–ITS2) of the nuclear ribosomal DNA in 119 specimens, representing 36 species of Stylosanthes and 7 species of the outgroup genera Arachis and Chapmannia. In all examined specimens of any particular diploid and (allo)polyploid species, only a single ITS sequence type was observed. This allowed us to identify a parental genome donor for some of the polyploids. In several diploid and polyploid species, different specimens contained a different ITS sequence. Some of these sequence types were present in more than one species. Parsimony analysis yielded several well-supported clades that agree largely with analyses of the chloroplast trnL intron and partially with the current sectional classification. Discordances between the nuclear and cpDNA analyses are explained by a process of allopolyploidization with inheritance of the cpDNA of one parent and fixation of the ITS sequences of the other. S. viscosa has been an important genome donor in this process of speciation by allopolyploidy. Received August 14, 2001; accepted March 4, 2002 Published online: November 14, 2002 Addresses of the authors: Jacqueline Vander Stappen, Steven Van Campenhout and Guido Volckaert (E-mail: guido.volckaert@agr.kuleuven.ac.be), Katholieke Universiteit Leuven, Laboratory of Gene Technology, Kasteelpark Arenberg 21, B-3001 Leuven, Belgium. Jan De Laet, American Museum of Natural History, Division of Invertebrate Zoology, Central Park West at 79th Street, New York 10024–5192, USA. Susana Gama-López, Universidad Nacional Autónoma de México, Unidad de Biología, Tecnología y Protipos (UBIPRO), FES-Iztacala, Laboratorio de Recursos Naturales, Av. de Los Barrios S/N, Colonia Los Reyes Iztacala, Municipio Tlalnepantla, Estado de México, C.P. 54090, México. Present address: Apartado Postal 154, Cto. Parque No. 3, C.P. 53102, México.     

Phylogenetic analysis and confirmation of the endospore-forming nature of Pasteuria penetrans based on the spo0A gene

Pasteuria penetrans is an obligate parasite of plant parasitic nematodes and has yet to be grown in vitro. We have cloned the pivotal sporulation gene, spo0A, which is the first whole gene yet to come from this organism. Partial spo0A sequences were also obtained from the related bacteria, Pasteuria ramosa and Alicyclobacillus acidocaldarius. Phylogenetic analyses using the spo0A sequence data from this and previous studies confirmed the closeness of the genera Pasteuria and members of the supergenus Bacillus. A segment of the spo0A gene was also used to show that genetic heterogeneity exists within and between populations of P. penetrans. This may explain, partly at least, the variability of P. penetrans as a biological control agent of nematodes.