Dataset Information

Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations.

ABSTRACT: BACKGROUND:The establishment of mutant populations together with the strategies for targeted mutation detection has been applied successfully to a large number of organisms including many species in the plant kingdom. Considerable efforts have been invested into research on tomato as a model for berry-fruit plants. With the progress of the tomato sequencing project, reverse genetics becomes an obvious and achievable goal. RESULTS:Here we describe the treatment of Solanum lycopersicum seeds with 1% EMS and the development of a new mutated tomato population. To increase targeted mutant detection throughput an automated seed DNA extraction has been combined with novel mutation detection platforms for TILLING in plants. We have adapted two techniques used in human genetic diagnostics: Conformation Sensitive Capillary Electrophoresis (CSCE) and High Resolution DNA Melting Analysis (HRM) to mutation screening in DNA pools. Classical TILLING involves critical and time consuming steps such as endonuclease digestion reactions and gel electrophoresis runs. Using CSCE or HRM, the only step required is a simple PCR before either capillary electrophoresis or DNA melting curve analysis. Here we describe the development of a mutant tomato population, the setting up of two polymorphism detection platforms for plants and the results of the first screens as mutation density in the populations and estimation of the false-positives rate when using HRM to screen DNA pools. CONCLUSION:These results demonstrate that CSCE and HRM are fast, affordable and sensitive techniques for mutation detection in DNA pools and therefore allow the rapid identification of new allelic variants in a mutant population. Results from the first screens indicate that the mutagen treatment has been effective with an average mutation detection rate per diploid genome of 1.36 mutation/kb/1000 lines.

SUBMITTER: Gady AL

PROVIDER: S-EPMC2763861 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations.

Gady Antoine Lf AL Hermans Freddy Wk FW Van de Wal Marion Hbj MH van Loo Eibertus N EN Visser Richard Gf RG Bachem Christian Wb CW

Plant methods 20091007

<h4>Background</h4>The establishment of mutant populations together with the strategies for targeted mutation detection has been applied successfully to a large number of organisms including many species in the plant kingdom. Considerable efforts have been invested into research on tomato as a model for berry-fruit plants. With the progress of the tomato sequencing project, reverse genetics becomes an obvious and achievable goal.<h4>Results</h4>Here we describe the treatment of Solanum lycopersi ...[more]

PMID: 19811648

Similar Datasets

Project description:BackgroundSmall peptides encoded as one- or two-exon genes in plants have recently been shown to affect multiple aspects of plant development, reproduction and defense responses. However, popular similarity search tools and gene prediction techniques generally fail to identify most members belonging to this class of genes. This is largely due to the high sequence divergence among family members and the limited availability of experimentally verified small peptides to use as training sets for homology search and ab initio prediction. Consequently, there is an urgent need for both experimental and computational studies in order to further advance the accurate prediction of small peptides.ResultsWe present here a homology-based gene prediction program to accurately predict small peptides at the genome level. Given a high-quality profile alignment, SPADA identifies and annotates nearly all family members in tested genomes with better performance than all general-purpose gene prediction programs surveyed. We find numerous mis-annotations in the current Arabidopsis thaliana and Medicago truncatula genome databases using SPADA, most of which have RNA-Seq expression support. We also show that SPADA works well on other classes of small secreted peptides in plants (e.g., self-incompatibility protein homologues) as well as non-secreted peptides outside the plant kingdom (e.g., the alpha-amanitin toxin gene family in the mushroom, Amanita bisporigera).ConclusionsSPADA is a free software tool that accurately identifies and predicts the gene structure for short peptides with one or two exons. SPADA is able to incorporate information from profile alignments into the model prediction process and makes use of it to score different candidate models. SPADA achieves high sensitivity and specificity in predicting small plant peptides such as the cysteine-rich peptide families. A systematic application of SPADA to other classes of small peptides by research communities will greatly improve the genome annotation of different protein families in public genome databases.

Project description:Background:Stroke is a leading cause of disability and the fifth leading cause of death in the USA. Intravenous alteplase is a highly effective clot-dissolving stroke treatment that must be given in a hospital setting within a time-sensitive window. To increase the use of intravenous alteplase in stroke patients, many US counties enacted policies mandating emergency medical service (EMS) paramedics to bypass local emergency departments and instead directly transport patients to specially equipped stroke centers. The objective of this mixed-methods study is to evaluate the effectiveness of policy enactment as an implementation strategy, how differences in policy structures and processes impact effectiveness, and to explore how the county, hospital, and policy factors explain variation in implementation and clinical outcomes. This paper provides a detailed description of an Agency for Healthcare Quality and Research (AHRQ)-funded protocol, including the use of the Consolidated Framework for Implementation Research (CFIR) in the qualitative design. Methods/design:We will construct the largest-ever national stroke database of Medicare enrollees (~ 1.5 million stroke patients) representing 896 policy counties paired with 1792 non-policy counties, then integrate patient-, hospital-, county-, and state-level covariates from eight different data sources. We will use a difference-in-differences analysis to estimate the overall effect of the policy enactment on intravenous alteplase use (implementation outcome) as well as key patient outcomes. We will also quantitatively examine if variation in the context (urban/rural status) and variation in policy features affect outcomes. Finally, a CFIR-informed multiple case study design will be used to interview informants in 72 stakeholders in 24 counties to identify and validate factors that enable policy effects. Discussion:Policies can be potent implementation strategies. However, the effects of EMS bypass policies to increase intravenous alteplase use have not been rigorously evaluated. By learning how context and policy structures impact alteplase implementation, as well as the barriers and facilitators experienced by stakeholders responsible for policy enactment, the results of this study will inform decisions regarding if and how EMS bypass policies should spread to non-policy counties, and if indicated, creation of a "best practices" toolkit.

Project description:INTRODUCTION:There is considerable interest in triaging victims of large vessel occlusion (LVO) strokes to comprehensive stroke centers. Timely access to interventional therapy has been linked to improved stroke outcomes. Accurate triage depends upon the use of a validated screening tool in addition to several emergency medical system (EMS)-specific factors. This study examines the integration of a modified Rapid Arterial oCcclusion Evaluation (mRACE) score into an existing stroke treatment protocol. METHODS:We performed a retrospective review of EMS and hospital charts of patients transported to a single comprehensive stroke center. Adult patients with an EMS provider impression of "stroke/TIA," "CVA," or "neurological problem" were included for analysis. EMS protocols mandated the use of the Cincinnati Prehospital Stroke Score (CPSS). The novel protocol authorized the use of the mRACE score to identify candidates for triage directly to the comprehensive stroke center. We calculated specificity and sensitivity for various stroke screens (CPSS and a mRACE exam) for the detection of LVO stroke. The score's metrics were evaluated as a surrogate marker for a successful EMS triage protocol. RESULTS:We included 312 prehospital charts in the final analysis. The CPSS score exhibited reliable sensitivity at 85%. Specificity of CPSS for an LVO was calculated at 73%. For an mRACE score of five or greater, the sensitivity was 25%. Specificity for mRACE was calculated at 75%. The positive predictive value of the mRACE score for an LVO was estimated at 12.50%. CONCLUSION:In this retrospective study of patients triaged to a single comprehensive stroke center, the addition of an LVO-specific screening tool failed to improve accuracy. Reliable triage of LVO strokes in the prehospital setting is a challenging task. In addition to statistical performance of a particular stroke score, a successful EMS protocol should consider system-based factors such as provider education and training. Study limitations can inform future iterations of LVO triage protocols.

Dataset Information

Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations.

Publications

Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets