Single Cell Genomics of Human Microbial Flora
人类微生物菌群的单细胞基因组学
基本信息
- 批准号:7628986
- 负责人:
- 金额:$ 38.63万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2005
- 资助国家:美国
- 起止时间:2005-05-12 至 2011-04-30
- 项目状态:已结题
- 来源:
- 关键词:AddressAdenoidal structureAdoptedAffectAllelesBacteriaBacterial GenomeBiochemical ReactionBiological AssayBiopsyBlood capillariesBody FluidsBorrelia burgdorferiCell SeparationCellsCharacteristicsChimera organismClinicalCollaborationsCollectionCommunicable DiseasesCommunitiesConfocal MicroscopyCytolysisDNADNA DamageDNA ProbesDNA SequenceDNA amplificationDNA biosynthesisDentistryDetectionDevelopmentDiagnosticDiagnostic testsDideoxy Chain Termination DNA SequencingDropsEcologyEnvironmentEnzymatic BiochemistryExanthemaFlow CytometryFluorescenceFreezingFundingGene RearrangementGeneral HospitalsGenomeGenomicsGenotypeGlassGoalsGrantHaemophilus influenzaeHealthcareHeatingHumanHuman bodyImageIn VitroInfectionInvestigationLabelLabyrinthLifeLocationLyme DiseaseMedicalMetagenomicsMethodsMicrobeMicrobial BiofilmsMicrobial GeneticsMicrofluidicsMicromanipulationMicroscopeMolecularMucous MembraneNeurologicNew JerseyOceansPathway interactionsPerformancePharyngeal structureProcessProtocols documentationPseudomonas aeruginosaPublishingReactionReagentResearchSalivaSamplingSkinSonicationSpecificitySpecimenStaphylococcus aureusStreptococcus pneumoniaeSurgical MeshSymptomsTechnologyTestingTicksTimeTissuesTwin Multiple BirthUniversitiesWashingtonWorkbrain tissuecapillarycell typechemical reactionclinical applicationfollow-upimmunocytochemistryimprovedlaser capture microdissectionmedical implantmethod developmentmicrobialmiddle earnanolitrenew technologynovelpathogenpoint-of-care diagnosticssample fixation
项目摘要
DESCRIPTION (provided by applicant): A new method called Multiple Displacement Amplification (MDA) has made it possible to sequence bacterial genomic DNA from a single cell. MDA is used to amplify the few femtograms of DNA in the cell for use in sequencing and for diagnostic and genotyping assays. MDA is rapidly being adopted for investigation of environmental bacterial communities, such as in oceans, where it can be used to discover novel microbes. However, there has been little effort yet to use MDA for studying bacteria living in the human body where powerful new research strategies could be developed to study the normal and pathogenic flora. Our objective is to develop the methods needed for use of MDA in amplifying bacterial genomes from human clinical specimens. Methods will be developed for tissues, fecal samples, body fluids and other specimen types for isolating single cells, lysing them to release their DNA, and carrying out MDA whole genome amplification. Preliminary studies have demonstrated high throughput isolation of Haemophilus influenzae from inner ear mucosa by flow cytometry. Isolation of single cells is also being carried out by micromanipulation in which a glass capillary is used under the microscope and specific bacteria are targeted by labeling with fluorescent DNA probes. Microfluidic MDA on chips is being developed for use in "point of care" diagnostic tests that could be used at any location without lab processing required. Laser capture microdissection (LCM) is being tested as an alternative method of cell isolation for use with infected tissues, particularly where bacterial biofilms or intracellular infection are suspected. Other pathogens to be studied will be Staphylococcus aureus, Streptococcus pneumoniae, and Pseudomonas aeruginosa from middle ear, and throat tissues and puss from wounds and infected medical implants. Ground braking methods are in development for combining confocal microscopy imaging of tissues and associated bacteria with single cell isolation and genomic sequencing. Bacteria from fecal samples will be used as part of a major project at Washington University in St. Louis to identify the microbes of the human gut. Borrelia burgdorferi, the cause of Lyme disease, has also been isolated from tick gut and DNA sequencing and genotyping demonstrated. Isolation from the characteristic Lyme disease rash in human skin is planned next and ultimately from human brain tissue where it is suspected of causing neurological symptoms. In parallel with the development of these new applications, our work in developing and optimizing the MDA method continues. Research is proposed into the basic enzymology of the MDA reaction and the chemical pathways involved in DNA amplification. Rapid progress is being made to improve the performance of MDA and to perfect methods and strategies for use of amplified DNA by both the Sanger sequencing method and 454 pyrosequencing. The overall goal is to complete the development of methods for in vitro amplification of genomic DNA from singles cells and to demonstrate the research applications of this new technology to the natural human bacterial flora and to infectious disease.
描述(由申请人提供):一种称为多重置换扩增(MDA)的新方法使从单个细胞中测序细菌基因组DNA成为可能。MDA用于扩增细胞中几个毫微微克的DNA,用于测序以及诊断和基因分型测定。MDA正迅速被用于环境细菌群落的研究,例如在海洋中,它可以用来发现新的微生物。然而,还没有什么努力使用MDA来研究生活在人体内的细菌,其中可以开发强大的新的研究策略来研究正常和致病植物群。我们的目标是开发使用MDA从人类临床标本中扩增细菌基因组所需的方法。将开发用于组织、粪便样本、体液和其他标本类型的方法,用于分离单细胞,裂解它们以释放它们的DNA,并进行MDA全基因组扩增。初步研究表明,通过流式细胞术从内耳粘膜中高通量分离流感嗜血杆菌。单细胞的分离也通过显微操作进行,其中在显微镜下使用玻璃毛细管,并通过用荧光DNA探针标记来靶向特定细菌。微流控MDA芯片正在开发中,用于“护理点”诊断测试,可以在任何位置使用,而无需实验室处理。激光捕获显微切割(LCM)正在测试作为一种替代方法的细胞分离用于感染的组织,特别是在细菌生物膜或细胞内感染的怀疑。其他要研究的病原体将是金黄色葡萄球菌,肺炎链球菌,和铜绿假单胞菌从中耳,咽喉组织和脓从伤口和感染的医疗植入物。地面制动方法正在开发中,用于将组织和相关细菌的共聚焦显微镜成像与单细胞分离和基因组测序相结合。来自粪便样本的细菌将被用作圣路易斯华盛顿大学一个重大项目的一部分,以识别人类肠道的微生物。莱姆病的病因伯氏疏螺旋体也已从蜱肠道中分离出来,并证明了DNA测序和基因分型。下一步计划从人类皮肤上的莱姆病皮疹特征中分离出来,最终从怀疑引起神经系统症状的人脑组织中分离出来。在开发这些新应用程序的同时,我们继续开发和优化MDA方法。研究提出了MDA反应的基本酶学和参与DNA扩增的化学途径。在改进MDA的性能和完善利用桑格测序法和454焦磷酸测序法扩增DNA的方法和策略方面正在取得快速进展。总体目标是完成从单个细胞中体外扩增基因组DNA的方法的开发,并展示这种新技术在自然人类细菌植物群和传染病中的研究应用。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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Roger Sender Lasken其他文献
Roger Sender Lasken的其他文献
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{{ truncateString('Roger Sender Lasken', 18)}}的其他基金
Transcriptomics from Single Circulating Tumor Cells
单个循环肿瘤细胞的转录组学
- 批准号:
7818833 - 财政年份:2009
- 资助金额:
$ 38.63万 - 项目类别:
Transcriptomics from Single Circulating Tumor Cells
单个循环肿瘤细胞的转录组学
- 批准号:
7940944 - 财政年份:2009
- 资助金额:
$ 38.63万 - 项目类别:
WHOLE GENOME AMPLIFICATION FOR RAPID LOW COST GENOTYPING
用于快速低成本基因分型的全基因组扩增
- 批准号:
7065215 - 财政年份:2005
- 资助金额:
$ 38.63万 - 项目类别:
WHOLE GENOME AMPLIFICATION FOR RAPID LOW COST GENOTYPING
用于快速低成本基因分型的全基因组扩增
- 批准号:
7287868 - 财政年份:2005
- 资助金额:
$ 38.63万 - 项目类别:
Single Cell Genomics of Human Microbial Flora
人类微生物菌群的单细胞基因组学
- 批准号:
7467152 - 财政年份:2005
- 资助金额:
$ 38.63万 - 项目类别:
Single Cell Genomics of Human Microbial Flora
人类微生物菌群的单细胞基因组学
- 批准号:
8463846 - 财政年份:2005
- 资助金额:
$ 38.63万 - 项目类别:
Single Cell Genomics of Human Microbial Flora
人类微生物菌群的单细胞基因组学
- 批准号:
8261090 - 财政年份:2005
- 资助金额:
$ 38.63万 - 项目类别:
WHOLE GENOME AMPLIFICATION FOR RAPID LOW COST GENOTYPING
用于快速低成本基因分型的全基因组扩增
- 批准号:
6908779 - 财政年份:2005
- 资助金额:
$ 38.63万 - 项目类别:
Single Cell Genomics of Human Microbial Flora
人类微生物菌群的单细胞基因组学
- 批准号:
8108682 - 财政年份:2005
- 资助金额:
$ 38.63万 - 项目类别:
WHOLE GENOME AMPLIFICATION FOR RAPID LOW COST GENOTYPING
用于快速低成本基因分型的全基因组扩增
- 批准号:
7291107 - 财政年份:2005
- 资助金额:
$ 38.63万 - 项目类别: