A novel paper MAP method for rapid high resolution histological analysis.
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ABSTRACT:
SUBMITTER: Lee M
PROVIDER: S-EPMC8639998 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: Molecular genetic alterations with prognostic significance have been described in childhood acute myeloid leukemia (AML). The aim of this study was to establish cost-effective techniques to detect mutations of FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), and a partial tandem duplication within the mixed-lineage leukemia (MLL-PTD) genes in childhood AML. PROCEDURE: Ninety-nine children with newly diagnosed AML were included in this study. We developed a fluorescent dye SYTO-82 based high-resolution melting (HRM) curve analysis to detect FLT3 internal tandem duplication (FLT3-ITD), FLT3 tyrosine kinase domain (FLT3-TKD), and NPM1 mutations. MLL-PTD was screened by real-time quantitative PCR. RESULTS: The HRM methodology correlated well with gold standard Sanger sequencing with less cost. Among the 99 patients studied, the FLT3-ITD mutation was associated with significantly worse event-free survival (EFS). Patients with the NPM1 mutation had significantly better EFS and overall survival. However, HRM was not sensitive enough for minimal residual disease monitoring. CONCLUSION: High-resolution melting was a rapid and efficient method for screening of FLT3 and NPM1 gene mutations. It was both affordable and accurate, especially in resource underprivileged regions. Our results indicated that HRM could be a useful clinical tool for rapid and cost-effective screening of the FLT3 and NPM1 mutations in AML patients.
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Project description:BACKGROUND:Genetic screens in Drosophila have provided a wealth of information about a variety of cellular and developmental processes. It is now possible to screen for mutant phenotypes in virtually any cell at any stage of development by performing clonal screens using the flp/FRT system. The rate-limiting step in the analysis of these mutants is often the identification of the mutated gene, however, because traditional mapping strategies rely mainly on genetic and cytological markers that are not easily linked to the molecular map. RESULTS:Here we describe the development of a single-nucleotide polymorphism (SNP) map for chromosome arm 3R. The map contains 73 polymorphisms between the standard FRT chromosome, and a mapping chromosome that carries several visible markers (rucuca), at an average density of one SNP per 370 kilobases (kb). Using this collection, we show that mutants can be mapped to a 400 kb interval in a single meiotic mapping cross, with only a few hundred SNP detection reactions. Discovery of further SNPs in the region of interest allows the mutation to be mapped with the same recombinants to a region of about 50 kb. CONCLUSION:The combined use of standard visible markers and molecular polymorphisms in a single mapping strategy greatly reduces both the time and cost of mapping mutations, because it requires at least four times fewer SNP detection reactions than a standard approach. The use of this map, or others developed along the same lines, will greatly facilitate the identification of the molecular lesions in mutants from clonal screens.
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Project description:This article describes FLASH (Fast Lithographic Activation of Sheets), a rapid method for laboratory prototyping of microfluidic devices in paper. Paper-based microfluidic devices are emerging as a new technology for applications in diagnostics for the developing world, where low cost and simplicity are essential. FLASH is based on photolithography, but requires only a UV lamp and a hotplate; no clean-room or special facilities are required (FLASH patterning can even be performed in sunlight if a UV lamp and hotplate are unavailable). The method provides channels in paper with dimensions as small as 200 microm in width and 70 microm in height; the height is defined by the thickness of the paper. Photomasks for patterning paper-based microfluidic devices can be printed using an ink-jet printer or photocopier, or drawn by hand using a waterproof black pen. FLASH provides a straightforward method for prototyping paper-based microfluidic devices in regions where the technological support for conventional photolithography is not available.
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Project description:Turning genes on and off is essential for development and homeostasis, yet little is known about the sequence and causal role of chromatin state changes during the repression of active genes. This is surprising, as defective gene silencing underlies developmental abnormalities and disease. Here we delineate the sequence and functional contribution of transcriptional repression mechanisms at high temporal resolution. Inducible entry of the NuRD-interacting transcriptional regulator Ikaros into mouse pre-B cell nuclei triggered immediate binding to target gene promoters. Rapid RNAP2 eviction, transcriptional shutdown, nucleosome invasion, and reduced transcriptional activator binding required chromatin remodeling by NuRD-associated Mi2beta/CHD4, but were independent of HDAC activity. Histone deacetylation occurred after transcriptional repression. Nevertheless, HDAC activity contributed to stable gene silencing. Hence, high resolution mapping of transcriptional repression reveals complex and interdependent mechanisms that underpin rapid transitions between transcriptional states, and elucidates the temporal order, functional role and mechanistic separation of NuRD-associated enzymatic activities.
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Project description:BackgroundComplex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs) from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolution melting (HRM) analysis is a new method in which polymerase chain reaction (PCR) and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB) is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB.MethodsHere, we used real time-PCR followed by HRM analysis to simultaneously identify several gene variations involved in inflammatory pathways on preterm labor. SNPs from TLR4, IL6, IL1 beta and IL12RB genes were analyzed in a case-control study. The results were confirmed either by sequencing or by PCR followed by restriction fragment length polymorphism.ResultsWe were able to simultaneously recognize the variations of four genes with similar accuracy than other methods. In order to obtain non-overlapping melting temperatures, the key step in this strategy was primer design. Genotypic frequencies found for each SNP are in concordance with those previously described in similar populations. None of the studied SNPs were associated with PTB.ConclusionsSeveral gene variations related to the same inflammatory pathway were screened through a new flexible, fast and non expensive method with the purpose of analyzing their association to PTB. It can easily be used for simultaneously analyze any set of SNPs, either as the first choice for new association studies or as a complement to large-scale genotyping analysis. Given that inflammatory pathway is in the base of several diseases, it is potentially useful to analyze a broad range of disorders.
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