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High molecular weight DNA purification instrument for metagenomics

用于宏基因组学的高分子量DNA纯化仪

基本信息

批准号：
7367898
负责人：
ANDRE MARZIALI
金额：
$ 20.58万
依托单位：
UNIVERSITY OF BRITISH COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-01 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7367898
关键词：
Address Antibiotics Bacterial DNA Biochemical Reaction Biological Markers Body Fluids Buffers Centrifugation Cloning Compatible Complex Coupled Cytolysis DNA DNA purification Development Electrodes Electrophoresis Environment Enzymes Escherichia coli Frequencies Future Gel Gene Cluster Generations Genomics Grant Injection of therapeutic agent Laboratories Left Length Libraries Licensing Malignant Neoplasms Mechanics Metagenomics Methods Molecular Weight Nature Nucleic Acids One-Step dentin bonding system Organism Pathogen detection Pathway interactions Performance Pharmaceutical Preparations Polymerase Chain Reaction Procedures Process Production RNA Degradation Range Reagent Recovery Research Personnel Resources Sampling Site Soil Solutions Speed Staging System Techniques Technology Temperature Testing Vacuum Work bacterial lysate base commercialization cost design desire electric field inhibitor/antagonist instrument instrumentation interest macromolecule microorganism next generation novel programs prototype reconstruction simulation size soil sampling tool

项目摘要

Less than 1% of all microorganisms observed in nature can be cultured in the laboratory, leaving researchers unable to study more than 99% of microorganisms in some environments - microorganisms that sometimes have unique abilities such as synthesis of compounds that could find use as new drugs or antibiotics. Metagenomics, the genomic reconstruction of unculturable microorganisms , is a powerful new tool for accessing the untapped resources of these organisms. In one approach, large fragments ofDMA from a sample containing unculturable microorganisms are extracted and cloned into a host such as E. coli to produce a metagenomics library. The library is then screened for utility of expressed compounds. Though new antibiotics and enzymes have been discovered with this method, successful production of compounds, such as antibiotics, synthesized by the original microorganism presents the difficult challenge of cloning fragments long enough to hold pathways encoded by gene clusters that are often over 100kb in length. Existing purification techniques tend to shear genomic DMAto fragments of 50kb or less. The discovery of a method to extract and purify high MW DMAfrom difficult samples such as soil will provide a breakthrough for metagenomics that may enable the discovery of many future drugs and antibiotics. We have recently demonstrated a non-linear electrophoretic method for DMA concentration that is capable of DMA manipulation and concentration without mechanical handling (such as centrifugation or pipetting). Extraction of DNA from bacterial lysate and 4,000 fold concentration factors have been demonstrated. This method is an excellent candidate for the next generation of methods for DNA extraction methods from complex samples such as soil or body fluids. We propose to develop an instrument to carry out this concentration method, validating its performance on extraction of high molecular weight DNA on soil samples for metagenomics studies aimed at discovery of new drugs and antibiotics. Licensing of this technology will be pursued to address other compelling applications including DNA extraction from body fluids for cancer biomarker and pathogen detection.

在自然界中观察到的所有微生物中，只有不到1%可以在实验室中培养，研究人员无法研究某些环境中超过99%的微生物-微生物，有时具有独特的能力，例如合成可用作新药的化合物，抗生素宏基因组学，即不可培养微生物的基因组重建，是一种强大的新技术。这是一个获取这些生物未开发资源的工具。在一种方法中，从含有不可培养微生物的样品中提取并克隆到宿主如E.杆菌来产生一个宏基因组文库。然后筛选文库中表达的化合物的效用。虽然用这种方法已经发现了新的抗生素和酶，但成功生产抗生素的方法仍然存在。由原始微生物合成的化合物，如抗生素，克隆片段的长度足以容纳由基因簇编码的途径，这些基因簇通常超过100 kb，长度现有的纯化技术倾向于将基因组DMA剪切成50 kb或更小的片段。从土壤等困难样品中提取和纯化高分子量DMA的方法的发现将提供这是宏基因组学的突破，可能会发现许多未来的药物和抗生素。我们最近已经证明了一种用于DMA浓度的非线性电泳方法，能够进行DMA操作和浓缩，而无需机械处理（如离心或移液）。从细菌裂解液中提取DNA和4,000倍浓缩因子已被证明是有效的。演示。这种方法是下一代DNA提取方法的一个很好的候选者方法从复杂的样品，如土壤或体液。我们建议开发一种仪器，通过实验验证了该方法提取土壤中高分子量DNA的效果用于宏基因组学研究的样本，旨在发现新药和抗生素。许可证，将继续研究其他引人注目的应用技术，包括从人体中提取DNA 用于癌症生物标志物和病原体检测的液体。