MOLECULAR GENETICS CORE

分子遗传学核心

• 批准号: 7699744
• 负责人: LOUIS M KUNKEL
• 金额: $ 19.2万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7699744
• 关键词: Affect; Area; Autistic Disorder; Bioinformatics; Biological Assay; Biomedical Research; Biometry; Blood capillaries; Brain Diseases; Child Support; Chromosome Mapping; Cloning; Complement; Complex; Computer software; Custom; DNA; DNA Resequencing; DNA Sequence; DNA Sequence Analysis; DNA Sequencing Facility; DNA purification; Data; Databases; Development; Developmental Disabilities; Dinucleotide Repeats; Disease; Disease Association; Dystrophin; Floor; Funding; Gene Expression; Genes; Genetic; Genetic Markers; Genome Mappings; Genomics; Genotype; Grant; Housing; Human; Human Genome Project; Individual; Label; Maps; Mental Retardation and Developmental Disabilities Research Centers; Messenger RNA; Methodology; Microarray Analysis; Microsatellite Repeats; Molecular; Molecular Genetics; Mutate; Mutation; Myopathy; Neurosciences; Ocular Motility Disorders; Outsourcing; Patients; Pattern; Pediatric Hospitals; Polymerase Chain Reaction; Probability; Program Research Project Grants; Publications; Recording of previous events; Research; Research Personnel; Research Project Grants; Resources; Sampling; Sequence Analysis; Services; Single Nucleotide Polymorphism; Speed; Statistical Data Interpretation; Statistically Significant; System; Techniques; Technology; Time; Tissues; Training; Update; Vendor; Work; base; capillary; cost; cost effective; design; experience; functional genomics; genetic linkage; genotyping technology; instrumentation; mRNA Expression; new technology; positional cloning; programs; research study; technology development; trait

6.a. 1.1. Introductory Overview Sequence databases and technology development that emanated from the Human Genome Project now offer avenues of molecular genetic and functional genomic analyses in biomedical research which were previously impossible as recently as the previous funding cycle for this Center Grant. The rapid pace of technological and instrumentation improvements that facilitate cutting edge molecular genetic and genomic approaches requires that we acquire new technologies, and have a highly skilled support staff that is adept at developing and supporting new instrumentation and methodologies. The Molecular Genetics Core has evolved to advance MRDDRC related research projects in three key areas: 1) high-throughput sequence analysis, 2) genomewide genetic marker genotype analysis for mapping and positional cloning, and 3) array-based transcriptional profiling. In providing these services to MRDDRC investigators, the Molecular Genetics Core has developed a cost-effective and efficient mechanism for these technically sophisticated approaches to be centrally managed to maintain quality and speed of data turn-around to MRDDRC investigators. The three functional components of the Molecular Genetics Core - Sequencing, Genotyping, and Expression Arrays - are all heavily utilized by MRDDRC investigators. Automated sequencing was established in the Core in the late 1980's following the cloning of the dystrophin locus, which ultimately led to the complete sequence analysis of three autosomal recessive forms of dystrophy. In its current form, the Core is being utilized in a high throughput sequencing strategy to identify mutations in all the known genes mutated in muscle diseases. Additionally, numerous MRDDRC investigators submit DMA for sequencing in the Core rather than outsourcing projects to commercial vendors because the in-house service is cost effective and rapid. Genotyping technologies supported by the Core include analysis of dinucleotide repeat (microsatellite) markers and single nucleotide polymorphisms (SNPs). These are performed as custom assays using the ABI 3730 capillary DMA analyzer for microsatellite markers, or on the ABI 7900 HT 384-well Real-time PCR machine for Taqman-based SNP genotyping. For whole genome mapping and genetic marker disease association studies, the Core supports microarray analysis of SNPs using both the Affymetrix 10k and 100k SNP genotyping arrays (which query 10,000 or 100,000 polymorphic human genetic markers respectively). In such studies, genomic DNA from affected and unaffected individuals is labeled and hybridized to the genotyping arrays. Using statistical analysis of the genotypes at each of the thousands of markers examined among the control and patient DNA samples, a probability of linkage between the disease trait and each genetic marker (SNP) can be assigned. The Core has supported MRDDRC investigators in mapping genes involved in eye movement disorders and identifying autosomal mutations involved in recessive disorders of brain development. The Affymetrix 10K and 100K SNP genotyping arrays are also being used to map other complex traits such as autism. The expression array component of the facility is an important new addition that has been used extensively by MRDDRC investigators to compare normal and diseased tissue mRNA expression patterns. In these studies, Affymetrix expression arrays are hybridized to probes generated from mRNA isolated from control and diseased tissues. Genes with statistically significant changes in expression (either up- or down-regulated) are identified, and form the basis for new hypothesis driven experiments to understand the molecular basis of the disease. Establishment of this technology was made possible by a Program Project grant to the Core Director. The array component of the Molecular Genetics Core is overseen by Drs. Isaac Kohane and Alan Beggs, and has extensive support from the Children's Hospital bioinformatics group led by Dr. Kohane. Close ties with this program are indicated by 14 collaborative publications between MRDDRC investigators and the bioinformatics group. Historically, the facility has tried to keep up with the latest technologic advances in sequencing, genotyping and expression arrays. Gene expression and SNP genotyping capabilities were recently upgraded with the purchase of the Illumina Bead Array system, which provides users access to customized arrays at lower cost than other platforms. The Core has increased sequencing capacity with the purchase of a second Applied Biosystems 48-capillary sequencer and further increased capacity by implementing an automated workstation for high throughput low volume DNA cycle-sequencing. The Core recently relocated to newly renovated and increased space on the 10th floor of the Enders research building. The staff is highly trained in all the methodologies used within the facility and continue to update their training as new techniques become available. The staff has extensive experience working with MRDDRC investigators in planning experiments and in the analysis of their data. In aggregate, this Core has a strong history and is essential to the research programs of MRDDRC investigators. 6.a.2. Overall Objective The overall objective of the Molecular Genetics Core is to provide high quality, low cost DNA sequencing, microsatellite genotyping and a variety of SNP genotyping and gene expression analysis technologies for MRDDRC investigators. The Core strives to complement these technologies with very strong biostatistics support. It also strives to serve as a reliable resource for investigators pursuing a broader understanding of the genetic basis of developmental disabilities.

6.a.1.1.简介概述 源于人类基因组计划的序列数据库和技术开发现在提供了 分子遗传学和功能基因组分析在生物医学研究中的途径 就像上一个资金周期一样，这笔中心拨款是不可能实现的。技术和技术的快速发展 促进尖端分子遗传和基因组方法的仪器改进需要 我们获得了新技术，并拥有一支技能高超的支持人员，他们擅长开发和 支持新的仪器和方法。分子遗传学的核心已经进化到 与MRDDRC相关的三个关键领域的研究项目：1)高通量序列分析，2)全基因组 用于定位和定位克隆的遗传标记基因分析，以及3)基于阵列的转录 侧写。在向MRDDRC调查人员提供这些服务的过程中，分子遗传学核心开发了一种 具有成本效益和高效的机制，以便对这些技术复杂的方法进行集中管理 以保持向MRDDRC调查人员提供数据的质量和速度。 分子遗传学核心的三个功能成分--测序、基因分型和表达 阵列--所有这些都被MRDDRC调查人员大量使用。在核心中建立了自动测序 在20世纪80年代末，S在克隆抗肌营养不良蛋白基因后，最终得出了完整的序列 三种常染色体隐性营养不良类型的分析。在其目前的形式中，核心正在被用于 高通量测序策略，以识别肌肉疾病中所有已知突变基因的突变。 此外，许多MRDDRC调查人员将DMA提交给核心进行排序，而不是外包 将项目提供给商业供应商，因为内部服务具有成本效益和速度快。 核心支持的基因分型技术包括分析二核苷酸重复(微卫星) 标记和单核苷酸多态性(SNPs)。这些都是使用ABI作为自定义分析执行的 3730毛细管DMA分析仪用于微卫星标记，或在ABI 7900 HT384-Well实时聚合酶链式反应 基于Taqman的SNP基因分型机器。用于全基因组作图和遗传标记疾病 关联研究，核心支持使用Affymetrix 10k和100k的SNPs微阵列分析 SNP基因分型阵列(分别查询10,000或100,000个多态人类遗传标记)。在……里面 这样的研究，来自受影响和未受影响的个体的基因组DNA被标记并与 基因分型阵列。使用对所检查的数千个标记中的每一个的基因类型的统计分析 在对照和患者DNA样本中，疾病特征和每个 可以指定遗传标记(SNP)。核心支持MRDDRC研究人员绘制基因图谱 参与眼球运动障碍并鉴定与隐性疾病有关的常染色体突变 大脑发育。Affymetrix 10K和100K SNP基因分型阵列也被用来定位其他 自闭症等复杂特征。 该工具的表达式数组组件是一个重要的新添加项，已被广泛使用 MRDDRC研究人员比较正常和病变组织的mRNA表达模式。在这些研究中， Affymetrix表达阵列与从对照和 有病的组织。在统计上有显著变化(上调或下调)的基因有 确定，并形成新的假说驱动的实验的基础，以了解 疾病。这项技术的建立是通过向Core提供的计划项目赠款实现的 导演。分子遗传学核心的阵列组件由Isaac Kohane博士和Alan博士监督 并得到了由Kohane博士领导的儿童医院生物信息学小组的广泛支持。关 与该项目的联系由MRDDRC调查人员与 生物信息学小组。 从历史上看，该设施一直试图跟上测序、基因分型等方面的最新技术进步 和表达式数组。基因表达和SNP基因分型能力最近随着 购买Illumina磁珠阵列系统，使用户能够以更低的成本访问定制的阵列 而不是其他平台。通过购买第二个应用程序，Core增加了测序能力 生物系统48-毛细管测序仪，并通过实施自动化工作站进一步提高容量 用于高通量、低容量的DNA循环测序。核心最近搬迁到新翻修的和 增加了Enders研究大楼10层的空间。工作人员在所有方面都受到了高度的培训 在设施内使用的方法，并随着新技术的出现继续更新其培训 可用。工作人员在与MRDDRC调查人员一起规划实验方面具有丰富的经验 并对他们的数据进行分析。总体而言，这一核心具有深厚的历史渊源，对研究至关重要 MRDDRC调查人员的计划。 6.A.2.总体目标 分子遗传学核心的总体目标是提供高质量、低成本的DNA测序， 微卫星基因分型和各种SNP基因分型和基因表达分析技术 MRDDRC调查人员。核心致力于用非常强大的生物统计学来补充这些技术 支持。它还努力成为调查人员寻求更广泛了解的可靠资源 发育障碍的遗传基础。