DIRECT SELECTION METHODS

直接选择法

• 批准号: 7468240
• 负责人: Michael Lovett
• 金额: $ 22.8万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-18 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7468240
• 关键词: Area; Biological; Birds; Collection; Complex; Conserved Sequence; DNA Sequence; Data; Disease; Elements; Emulsions; Genome; Genomic Library; Genomics; Human; Human Genetics; Indium; Individual; Length; Methods; Morphogenesis; Nucleic Acids; Phenols; Polymerase Chain Reaction; Range; Rate; Repetitive Sequence; Research Personnel; Speed; Testing; Time; comparative; craniofacial; interest; positional cloning; research study

DESCRIPTION (provided by applicant): In this R21 application we propose to implement improvements to direct genomic selection to more rapidly capture collections of highly conserved sequence elements across diverse genomes and diverse species. We shall also implement improvements that are more targeted towards the non-conserved sequences that adjoin conserved elements, in order to capture a more complete coverage of orthologous regions between individuals and species. These methodological improvements should have broad relevance and interest to many areas of human genetics and comparative genomics. To increase the speed in which direct selection experiments can be performed. The most time consuming aspect of the current selection method is the length of hybridization time required to reach a Cot1/2 of 25 to 100. Most investigators would prefer this to be simplified and standardized into a "kit-like" format. We shall test phenol emulsion methods to increase nucleic acid reassociation rates and to standardize the selection parameters. To explore methods for enhancing the coverage of direct selection. We shall extend the sequence coverage of direct selection by standardizing random shearing methods to generate larger fragment lengths. We shall employ these, along with long-range PCR methods, to capture more of the non-conserved or repetitive sequences across large contigs. We shall also construct and use biologically amplifiable, singlestranded libraries of genomic DNAs from which coverage can be extended in a secondary selection step. To increase the target genomic interval for selections to greater than 1Mb. Many largescale positional cloning projects, whether for "simple" or complex diseases, result in the identification of large genomic target intervals (~1Mb). Likewise, many cross-species comparative genomics projects would benefit from the ability to target large regions. Our preliminary data indicate that 470kb can be efficiently employed as a target for direct selection. We shall demonstrate proof of principle that >1Mb can be efficiently used in these methods. To demonstrate the feasibility of conducting cross-species direct selections on a ~1Mb interval in 20 evolutionarily diverged species. Comparative genomics is critically dependent on obtaining large scale genomic DNA sequence information across diverse species. We shall apply selection methods to a genomic region that has biological relevance to craniofacial morphogenesis in many species (including humans). We shall employ targeted selections (and a limited amount of comparative DNA sequencing) across the Bmp4 locus in avian species that had last common ancestors between 40 million and 120 million years ago.

描述（由申请人提供）：在此 R21 申请中，我们建议实施改进以直接基因组选择，以更快地捕获跨不同基因组和不同物种的高度保守的序列元件的集合。我们还将实施更针对与保守元件相邻的非保守序列的改进，以便更完整地覆盖个体和物种之间的直系同源区域。这些方法学的改进应该与人类遗传学和比较基因组学的许多领域具有广泛的相关性和兴趣。 提高直接选择实验的执行速度。最 当前选择方法的一个耗时方面是达到 25 至 100 的 Cot1/2 所需的杂交时间长度。大多数研究人员希望将其简化并标准化为“类似试剂盒”的格式。我们将测试苯酚乳剂方法以提高核酸重联率并标准化选择参数。 探索提高直选覆盖率的方法。我们将通过标准化随机剪切方法来扩展直接选择的序列覆盖范围，以生成更大的片段长度。我们将使用这些方法以及长程 PCR 方法来捕获大型重叠群中更多的非保守或重复序列。我们还将构建和使用可生物扩增的单链基因组 DNA 文库，可以在二次选择步骤中扩展覆盖范围。将选择的目标基因组间隔增加到大于 1Mb。许多大规模的定位克隆项目，无论是针对“简单”还是复杂的疾病，都会导致大基因组目标区间 (~1Mb) 的鉴定。同样，许多跨物种比较基因组学项目将受益于针对大区域的能力。我们的初步数据表明，470kb 可以有效地用作直接选择的目标。我们将证明原理证明 >1Mb 可以在这些方法中有效使用。 证明在 20 个进化分化的物种中以约 1Mb 的间隔进行跨物种直接选择的可行性。比较基因组学关键依赖于获得大量 扩展不同物种的基因组 DNA 序列信息。我们将应用选择方法到与许多物种（包括人类）的颅面形态发生具有生物学相关性的基因组区域。我们将对在 4000 万至 1.2 亿年前拥有最后共同祖先的鸟类物种的 Bmp4 基因座进行有针对性的选择（以及有限数量的比较 DNA 测序）。