MOLECULAR GENETICS CORE

6.a. 1.1.介绍性概述 人类基因组计划所产生的序列数据库和技术发展现在提供了 生物医学研究中的分子遗传学和功能基因组分析的途径， 这是不可能的，因为最近为这个中心赠款的前一个资助周期。技术的快速发展， 促进尖端分子遗传学和基因组方法的仪器改进需要 我们获得新技术，并拥有一个高度熟练的支持人员，善于开发和 支持新的工具和方法。分子遗传学核心已经发展到 MRDDRC的相关研究项目有三个关键领域：1）高通量序列分析，2）全基因组 用于作图和定位克隆的遗传标记基因型分析，和3）基于阵列的转录 侧写在为MRDDRC研究人员提供这些服务时，分子遗传学核心已经开发了一个 对这些技术复杂的方法进行集中管理的具有成本效益和效率的机制 保持向MRDDRC研究者提供数据的质量和速度。 分子遗传学核心的三个功能组件-测序、基因分型和表达 阵列-MRDDRC调查人员大量使用。在核心区建立了自动测序 在20世纪80年代后期，随着抗肌萎缩蛋白基因座的克隆，最终导致了完整的序列， 分析三种常染色体隐性形式的营养不良。在其目前的形式，核心正在利用一个 高通量测序策略，以确定在肌肉疾病中突变的所有已知基因的突变。 此外，许多MRDDRC研究者提交DMA用于核心测序，而不是外包 由于内部服务具有成本效益和快速性， 核心支持的基因分型技术包括二核苷酸重复序列（微卫星）分析 标记和单核苷酸多态性（SNP）。这些是使用ABI作为自定义测定进行的 微卫星标记的3730毛细管DMA分析仪，或ABI 7900 HT 384孔实时PCR 用于基于Taqman的SNP基因分型的机器。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Affyss 10 k和100 k的SNP微阵列分析 SNP基因分型阵列（分别查询10，000或100，000个多态性人类遗传标记）。在 在这些研究中，来自受影响和未受影响个体的基因组DNA被标记并与来自受影响个体的基因组DNA杂交。 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型的统计分析， 在对照和患者DNA样品中，疾病性状与每个基因之间的连锁概率是 遗传标记（SNP）。核心支持MRDDRC研究人员绘制基因图谱 参与眼球运动障碍和识别常染色体突变参与隐性疾病， 大脑发育Affysses 10 K和100 K SNP基因分型阵列也被用于绘制其他 复杂的特征，如自闭症。 该工具的表达式数组组件是一个重要的新增组件， MRDDRC研究人员比较正常和患病组织mRNA表达模式。在这些研究中， 亲和素表达阵列与从分离自对照的mRNA产生的探针杂交， 病变组织具有统计学显著表达变化（上调或下调）的基因是 确定，并形成新的假设驱动的实验，以了解的分子基础， 疾病这项技术的建立是由一项计划项目赠款的核心 导演分子遗传学核心的阵列组件由Isaac Kohane博士和Alan Beggs，并得到了Kohane博士领导的儿童医院生物信息学小组的广泛支持。密切 与该计划的联系由MRDDRC调查人员和 生物信息学组。 从历史上看，该设施一直试图跟上最新的技术进步，在测序，基因分型， 表达式数组。基因表达和SNP基因分型能力最近升级， 购买Illumina Bead Array系统，使用户能够以较低的成本获得定制阵列 相比其他平台。核心增加了测序能力与购买第二个应用 Biosystems 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 用于高通量低体积DNA循环测序。核心最近搬迁到新装修， 恩德斯研究大楼10楼的空间增加了。工作人员在所有方面都训练有素。 在设施内使用的方法，并继续更新他们的培训，因为新技术成为 available.工作人员在规划实验方面与MRDDRC研究人员合作拥有丰富的经验 以及对数据的分析。总的来说，这个核心有着悠久的历史，对研究至关重要。 MRDDRC研究人员的计划。 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。核心努力用非常强大的生物统计学来补充这些技术 支持.它还努力为调查人员提供可靠的资源，以便更广泛地了解 发育障碍的遗传基础

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.