The BiSearch web server

BACKGROUND: A large number of PCR primer-design softwares are available online. However, only very few of them can be used for the design of primers to amplify bisulfite-treated DNA templates, necessary to determine genomic DNA methylation profiles. Indeed, the number of studies on bisulfite-treated templates exponentially increases as determining DNA methylation becomes more important in the diagnosis of cancers. Bisulfite-treated DNA is difficult to amplify since undesired PCR products are often amplified due to the increased sequence redundancy after the chemical conversion. In order to increase the efficiency of PCR primer-design, we have developed BiSearch web server, an online primer-design tool for both bisulfite-treated and native DNA templates. RESULTS: The web tool is composed of a primer-design and an electronic PCR (ePCR) algorithm. The completely reformulated ePCR module detects potential mispriming sites as well as undesired PCR products on both cDNA and native or bisulfite-treated genomic DNA libraries. Due to the new algorithm of the current version, the ePCR module became approximately hundred times faster than the previous one and gave the best performance when compared to other web based tools. This high-speed ePCR analysis made possible the development of the new option of high-throughput primer screening. BiSearch web server can be used for academic researchers at the site. CONCLUSION: BiSearch web server is a useful tool for primer-design for any DNA template and especially for bisulfite-treated genomes. The ePCR tool for fast detection of mispriming sites and alternative PCR products in cDNA libraries and native or bisulfite-treated genomes are the unique features of the new version of BiSearch software

Repeats as global DNA methylation marker in bovine preimplantation embryos

DNA methylation undergoes dynamic changes and is a crucial part of the epigenetic regulation during mammalian early development. To determine the DNA methylation levels in bovine embryos, we applied a bisulfite sequencing based method aimed at repetitive sequences including three retrotransposons (L1_BT, BovB, and ERV1-1-I_BT) and Satellite I. A more accurate estimate of the global DNA methylation level compared to previous methods using only one repeat sequence, like Alu, could be made by calculation of the weighted arithmetic mean of multiple repetitive sequences, considering the copy number of each repetitive sequence. Satellite I and L1_BT showed significant methylation reduction at the blastocyst stage, while BovB and ERV1-1-I_BT showed no difference. The mean methylation level of the repetitive sequences during preimplantation development was the lowest at the blastocyst stage. No methylation difference was found between embryos cultured in 5% and 20% O-2. Because mutations of CpGs negatively influence the calculation accuracy, we checked the mutation rate of the sequenced CpG sites. Satellite I and L1_BT showed a relatively low mutation rate (1.92 and 3.72% respectively) while that of ERV1-1-I_BT and BovB was higher (11.95 and 24% respectively). Therefore we suggest using a combination of repeats with low mutation rate, taking into account the proportion of each sequence, as a relatively quick marker for the global DNA methylation status of preimplantation stages and possibly also for other cell types

Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis

AbstractThe polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator

Improved assay-dependent searching of nucleic acid sequence databases

Nucleic acid-based biochemical assays are crucial to modern biology. Key applications, such as detection of bacterial, viral and fungal pathogens, require detailed knowledge of assay sensitivity and specificity to obtain reliable results. Improved methods to predict assay performance are needed for exploiting the exponentially growing amount of DNA sequence data and for reducing the experimental effort required to develop robust detection assays. Toward this goal, we present an algorithm for the calculation of sequence similarity based on DNA thermodynamics. In our approach, search queries consist of one to three oligonucleotide sequences representing either a hybridization probe, a pair of Padlock probes or a pair of PCR primers with an optional TaqMan™ probe (i.e. in silico or ‘virtual’ PCR). Matches are reported if the query and target satisfy both the thermodynamics of the assay (binding at a specified hybridization temperature and/or change in free energy) and the relevant biological constraints (assay sequences binding to the correct target duplex strands in the required orientations). The sensitivity and specificity of our method is evaluated by comparing predicted to known sequence tagged sites in the human genome. Free energy is shown to be a more sensitive and specific match criterion than hybridization temperature

Methylation-specific PCR: four steps in primer design

Methylation-specific PCR (MSP) is still the method of choice for a single gene methylation study. The proper design of the primer pairs is a prerequisite for obtaining reliable PCR results. Despite numerous protocols describing the rules for MSP primer design, none of them provide a comprehensive approach to the problem. Our aim was to depict a workflow for the primer design that is concise and easy to follow. In order to achieve this goal, adequate tools for promoter sequence retrieval, MSP primer design and subsequent in silico analysis are presented and discussed. Furthermore, a few instructive examples regarding a good versus a poor primer design are provided. Finally, primer design is demonstrated according to the proposed workflow. This article aims to provide researchers, interested in a single gene methylation studies, with useful information regarding successful primer design

Long non-coding RNA expression profiling in the NCI60 cancer cell line panel using high-throughput RT-qPCR

Long non-coding RNAs (lncRNAs) form a new class of RNA molecules implicated in various aspects of protein coding gene expression regulation. To study lncRNAs in cancer, we generated expression profiles for 1707 human lncRNAs in the NCI60 cancer cell line panel using a high-throughput nanowell RT-qPCR platform. We describe how qPCR assays were designed and validated and provide processed and normalized expression data for further analysis. Data quality is demonstrated by matching the lncRNA expression profiles with phenotypic and genomic characteristics of the cancer cell lines. This data set can be integrated with publicly available omics and pharmacological data sets to uncover novel associations between lncRNA expression and mRNA expression, miRNA expression, DNA copy number, protein coding gene mutation status or drug response

Genome-wide methylome analysis using MethylCap-seq uncovers 4 hypermethylated markers with high sensitivity for both adeno- and squamous-cell cervical carcinoma

Background: Cytology-based screening methods for cervical adenocarcinoma (ADC) and to a lesser extent squamous-cell carcinoma (SCC) suffer from low sensitivity. DNA hypermethylation analysis in cervical scrapings may improve detection of SCC, but few methylation markers have been described for ADC. We aimed to identify novel methylation markers for the early detection of both ADC and SCC. Results: Genome-wide methylation profiling for 20 normal cervices, 6 ADC and 6 SCC using MethylCap-seq yielded 53 candidate regions hypermethylated in both ADC and SCC. Verification and independent validation of the 15 most significant regions revealed 5 markers with differential methylation between 17 normals and 13 cancers. Quantitative methylation-specific PCR on cervical cancer scrapings resulted in detection rates ranging between 80% and 92% while between 94% and 99% of control scrapings tested negative. Four markers (SLC6A5, SOX1, SOX14 and TBX20) detected ADC and SCC with similar sensitivity. In scrapings from women referred with an abnormal smear (n = 229), CIN3+ sensitivity was between 36% and 71%, while between 71% and 93% of adenocarcinoma in situ (AdCIS) were detected; and CIN0/1 specificity was between 88% and 98%. Compared to hrHPV, the combination SOX1/SOX14 showed a similar CIN3+ sensitivity (80% vs. 75%, respectively, P>0.2), while specificity improved (42% vs. 84%, respectively, P < 10(-5)). Conclusion: SOX1 and SOX14 are methylation biomarkers applicable for screening of all cervical cancer types

Methylation of migraine-related genes in different tissues of the rat

17β-Estradiol, an epigenetic modulator, is involved in the increased prevalence of migraine in women. Together with the prophylactic efficacy of valproate, which influences DNA methylation and histone modification, this points to the involvement of epigenetic mechanisms. Epigenetic studies are often performed on leukocytes, but it is unclear to what extent methylation is similar in other tissues. Therefore, we investigated methylation of migraine-related genes that might be epigenetically regulated (CGRP-ergic pathway, estrogen receptors, endothelial NOS, as well as MTHFR) in different migraine-related tissues and compared this to methylation in rat as well as human leukocytes. Further, we studied whether 17β-estradiol has a prominent role in methylation of these genes. Female rats (n = 35) were ovariectomized or shamoperated and treated with 17b-estradiol or placebo. DNA was isolated and methylation was assessed through bisulphite treatment and mass spectrometry. Human methylation data were obtained using the Illumina 450k genome-wide methylation array in 395 female subjects from a population-based cohort study. We showed that methylation of the Crcp, Calcrl, Esr1 and Nos3 genes is tissue-specific and that methylation in leukocytes was not correlated to that in other tissues. Interestingly, the interindividual variation in methylation differed considerably between genes and tissues. Furthermore we showed that methylation in human leukocytes was similar to that in rat leukocytes in our genes of interest, suggesting that rat may be a good model to study human DNA methylation in tissues that are difficult to obtain. In none of the genes a significant effect of estradiol treatment was observed

CADM1 is a strong neuroblastoma candidate gene that maps within a 3.72 Mb critical region of loss on 11q23

<p>Abstract</p> <p>Background</p> <p>Recurrent loss of part of the long arm of chromosome 11 is a well established hallmark of a subtype of aggressive neuroblastomas. Despite intensive mapping efforts to localize the culprit 11q tumour suppressor gene, this search has been unsuccessful thus far as no sufficiently small critical region could be delineated for selection of candidate genes.</p> <p>Methods</p> <p>To refine the critical region of 11q loss, the chromosome 11 status of 100 primary neuroblastoma tumours and 29 cell lines was analyzed using a BAC array containing a chromosome 11 tiling path. For the genes mapping within our refined region of loss, meta-analysis on published neuroblastoma mRNA gene expression datasets was performed for candidate gene selection. The DNA methylation status of the resulting candidate gene was determined using re-expression experiments by treatment of neuroblastoma cells with the demethylating agent 5-aza-2'-deoxycytidine and bisulphite sequencing.</p> <p>Results</p> <p>Two small critical regions of loss within 11q23 at chromosomal band 11q23.1-q23.2 (1.79 Mb) and 11q23.2-q23.3 (3.72 Mb) were identified. In a first step towards further selection of candidate neuroblastoma tumour suppressor genes, we performed a meta-analysis on published expression profiles of 692 neuroblastoma tumours. Integration of the resulting candidate gene list with expression data of neuroblastoma progenitor cells pinpointed <it>CADM1 </it>as a compelling candidate gene. Meta-analysis indicated that <it>CADM1 </it>expression has prognostic significance and differential expression for the gene was noted in unfavourable neuroblastoma versus normal neuroblasts. Methylation analysis provided no evidence for a two-hit mechanism in 11q deleted cell lines.</p> <p>Conclusion</p> <p>Our study puts <it>CADM1 </it>forward as a strong candidate neuroblastoma suppressor gene. Further functional studies are warranted to elucidate the role of <it>CADM1 </it>in neuroblastoma development and to investigate the possibility of <it>CADM1 </it>haploinsufficiency in neuroblastoma.</p