Nesta página, faça o download de artigos interessantes que descrevem e dão exemplos de outras aplicações que você pode fazer com seu aparelho sequenciador de DNA. Vá além do sequenciamento e da análise de microssatélites.
Artigo de um grupo do Jardim Botânico do Rio de Janeiro, demonstrando o uso da técnica de MSAP para análise do perfil de metilação em plantas
Abstract: Background: Epigenetic modifications, such as cytosine methylation, are inherited in plant species and may occur in response to biotic or abiotic stress, affecting gene expression without changing genome sequence. Laguncularia racemosa, a mangrove species, occurs in naturally contrasting habitats where it is subjected daily to salinity and nutrient variations leading to morphological differences. This work aims at unraveling how CpG-methylation variation is distributed among individuals from two nearby habitats, at a riverside (RS) or near a salt marsh (SM), with different environmental pressures and how this variation is correlated with the observed morphological variation.
Principal Findings: Significant differences were observed in morphological traits such as tree height, tree diameter, leaf width and leaf area between plants from RS and SM locations, resulting in smaller plants and smaller leaf size in SM plants. Methyl-Sensitive Amplified Polymorphism (MSAP) was used to assess genetic and epigenetic (CpG-methylation) variation in L. racemosa genomes from these populations. SM plants were hypomethylated (14.6% of loci had methylated samples) in comparison to RS (32.1% of loci had methylated samples). Within-population diversity was significantly greater for epigenetic than genetic data in both locations, but SM also had less epigenetic diversity than RS. Frequency-based (GST) and
multivariate (bST) methods that estimate population structure showed significantly greater differentiation among locations for epigenetic than genetic data. Co-Inertia analysis, exploring jointly the genetic and epigenetic data, showed that individuals with similar genetic profiles presented divergent epigenetic profiles that were characteristic of the population in a particular environment, suggesting that CpG-methylation changes may be associated with environmental heterogeneity.
Conclusions: In spite of significant morphological dissimilarities, individuals of L. racemosa from salt marsh and riverside presented little genetic but abundant DNA methylation differentiation, suggesting that epigenetic variation in natural plant populations has an important role in helping individuals to cope with different environments.
Exemplo de uso da técnica de PCR seguida por restrição terminal dos fragmentos e análise em por eletroforese capilar para estudos de diversidade genética
Abstract: T-RFLP analysis is a technique used to study complex microbial communities based on variation in the 16S rRNA gene. T-RFLP analysis can be used to examine microbial community structure and community dynamics in response to changes in different environmental parameters or to study bacterial populations in natural habitats. It has been applied to the study of complex microbial communities in diverse environments such as soil, marine and activated sludge systems as well as in a study to characterize oral bacterial flora in saliva in healthy subjects versus patients with periodontitis. The T-RFLP technique is a cultureindependent, rapid, sensitive and reproducible method of assessing diversity of complex communities without the need for any genomic sequence information. The technique provides information on a collection of microorganisms that may be present in a given sample.
Introduction: As an Amplified Fragment Length Polymorphism (AFLP®)-based gene expression profiling method, the HiCEP method requires no prior sequence information and has a reduced rate of false positives with a high degree of detection of both coding and noncoding transcripts. After HiCEP analysis, fragments of interest can be purified and cloned from agarose gels, and sequenced to identify the transcripts. If whole genome sequence information for the organism under study is known, the fragments of interest can be identified by bioinformatic prediction using the sequence information available from public databases
and the restriction enzyme recognition sites used in the HiCEP workflow.
Uso da técnica de Análise de Fragmentos para avaliação de expressão gênica em larga escala.
Abstract: We have developed an AFLP-based gene expression profiling method called `high coverage expression profiling' (HiCEP) analysis. By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to ~4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased. As a result we can determine the relationship between peaks and original transcripts unequivocally. This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks. This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species. The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold in gene expression. Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for
fingerprinting mRNAs. Therefore, the method detects 70±80% of all transcripts, including noncoding transcripts, unknown and known genes.Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available.
Abstract: The use of capillary electrophoresis with fluorescently labeled nucleic acids revolutionized DNA sequencing, effectively fueling the genomic revolution. We present an application of this technology for the high-throughput structural analysis of nucleic acids by chemical and enzymatic mapping (‘footprinting’). We achieve the throughput and data quality necessary for genomic-scale structural analysis by combining fluorophore labeling of nucleic acids with novel quantitation algorithms. We implemented these algorithms in the CAFA (capillary automated footprinting analysis) open-source software that is downloadable gratis from https://simtk.org/home/cafa. The accuracy, throughput and reproducibility of CAFA analysis are demonstrated using hydroxyl radical footprinting of RNA. The versatility of CAFA is illustrated by dimethyl
sulfate mapping of RNA secondary structure and DNase I mapping of a protein binding to a specific
sequence of DNA. Our experimental and computational approach facilitates the acquisition of highthroughput
chemical probing data for solution structural analysis of nucleic acids.
Aplication note que descreve a técnica de AFLP, suas aplicações e vantagens.
Introduction
Amplified fragment length polymorphism (AFLP®) analysis is a genetic mapping technique that uses selective
amplification of a subset of restriction enzyme-digested DNA fragments to generate a unique fingerprint for a
particular genome. First developed for plant studies, AFLP analysis is used for a variety of applications, such as:
• Creation of genetic maps for new species;
• Determination of relatedness among cultivars;
• Establishment of linkage groups in crosses;
• Genetic diversity and molecular phylogeny studies.
Abstract: Annotated DNA samples that had been previously analyzed were tested using multiplex ligation-dependent probe amplification (MLPA) assays containing probes targeting BRCA1, BRCA2, and MMR (MLH1/MSH2 genes) and the 9p21 chromosomal region. MLPA polymerase chain reaction products were separated on a capillary electrophoresis platform, and the data were analyzed using GeneMapper v4.0 software (Applied Biosystems, Foster City, CA). After signal normalization, loci regions that had undergone deletions or duplications were identified using the GeneMapper Report Manager and verified using the DyeScale functionality. The results highlight an easy-to-use, optimal sample preparation and analysis workflow that can be used for both small- and large-scale studies.
Abstract: Here we provide a detailed protocol for the analysis of protein-linked glycans on DNA sequencing equipment. This protocol satisfies the glyco-analytical needs of many projects and can form the basis of ‘glycomics’ studies, in which robustness, high throughput, high sensitivity and reliable quantification are of paramount importance. The protocol routinely resolves isobaric glycan stereoisomers, which is much more difficult by mass spectrometry (MS). Earlier methods made use of polyacrylamide gel–based sequencers, but we have now adapted the technique to multicapillary DNA sequencers, which represent the state of the art today. In addition, we have integrated an option for HPLC-based fractionation of highly anionic 8-amino-1,3,6-pyrenetrisulfonic acid (APTS)-labeled glycans before rapid capillary electrophoretic profiling. This option facilitates either two-dimensional profiling of complex glycan mixtures and exoglycosidase sequencing, or MS analysis of particular compounds of interest rather than of the total pool of glycans in a sample.
Abstract: Heteroduplex analysis (HA) has proven to be a robust tool for mutation detection. HA by capillary array electrophoresis (HA-CAE) was developed to increase throughput and allow the scanning of large multiexon genes in multicapillary DNA sequencers. HA-CAE is a straightforward and high-throughput technique to detect both known and novel DNA variants with a high level of sensitivity and specificity. It consists of only three steps: multiplex-PCR using fluorescently labeled primers, heteroduplex formation and electrophoresis in a multicapillary DNA sequencer. It allows, e.g., the complete coding and flanking intronic sequences of BRCA1 and BRCA2 genes from two patients (approximately 25 kb each) to be scanned in a single run of a 16-capillary sequencer, and has enabled us to detect 150 different mutations to date (both single nucleotide substitutions, or SNSs, and small insertions/deletions). Here, we describe the protocol developed in our laboratory to scan BRCA1, BRCA2, MLH1, MSH2 and MSH6 genes using an ABI3130XL sequencer. This protocol could be adapted to other instruments or to the study of other large multiexon genes and can be completed in 7–8 h.
Relative Fluorescent Quantitation on Capillary Electrophoresis - Screening for Loss of Heterozygosity in Tumor Samples.pdf
Introduction
A variety of capillary electrophoresisbased fragment analysis applications require peak-height comparisons across samples as a relative quantitation method. Some of these applications include screening for loss of heterozygosity using microsatellites or Single Nucleotide Polymorphisms (SNPs), aneuploidy assays, and detection of large chromosomal deletions. The success of relative fluorescent quantitation depends on having optimized assays, a robust and sensitive capillary electrophoresis (CE) platform, and accurate analysis software.
Relative Fluorescent Quantitation on Capillary Electrophoresis - Screening for Loss of Heterozygosity in Tumor Samples.pdf
Introduction
A variety of capillary electrophoresisbased fragment analysis applications require peak-height comparisons across samples as a relative quantitation method. Some of these applications include screening for loss of heterozygosity using microsatellites or Single Nucleotide Polymorphisms (SNPs), aneuploidy assays, and detection of large chromosomal deletions. The success of relative fluorescent quantitation depends on having optimized assays, a robust and sensitive capillary electrophoresis (CE) platform, and accurate analysis software.
