Automated preparation of megabase genomic DNA

描述（由申请人提供）：项目摘要/摘要：a）我们建议开发一种经济的自动化系统（BioSprint 96），用于从血液或口腔拭子中制备亚兆碱基（250- 500 kb），优选兆碱基大小的基因组DNA。B）相同系统上的类似方案将使得能够通过单倍型特异性提取（HSE）靶向提取特异性的单倍体基因区域。该项目将导致在现有仪器上开发新的DNA样品制备方案，与现有方法相比，大大提高了提取DNA的长度。所提出的方法预计将显着更快，更符合成本效益比现有的方法分离的高分子量DNA和样品制备的个人单倍型。该项目的完成将产生一种通用的DNA分离和分子单体型制备技术，广泛适用于生物医学研究，如关联研究、疾病预后、药物基因组学、肿瘤学检测和一般的基因组学。我们将使用特异性探针和磁珠将亚巨噬细胞和巨噬细胞二倍体基因组DNA分离成其基于签名序列和小至单核苷酸（SNP）的多态性组分的单倍体组分。这些极长链、单倍体基因组DNA制备物然后可以直接用于常规基因分型过程，例如实时PCR、测序或基于阵列的SNP分析，以确定长范围分子单倍型数据。我们将为多达3种常见应用开发套件。项目叙述：目前分离大DNA片段的项目的目标是产生未改变的基因组DNA的延伸的单倍体基因区域。由于该方法适用于个体患者的未扩增的基因组DNA，因此它保留了DNA片段的原始状态，例如保留了其表观遗传甲基化模式，并且不需要统计推断来推导单倍型。这将提供一种改进的工具来表征用于器官移植的潜在复杂的遗传特征（例如主要组织相容性复合物（MHC）和杀伤免疫球蛋白样受体（KIR）基因座）和遗传性疾病。通过将大区域的DNA分离并分离到其亲本部分中，每个亲本贡献的独特遗传差异可以用于尝试将复杂的遗传疾病与特定的等位基因组合或单倍型联系起来。这一认识应导致更好地了解遗传性疾病的病因，这最终应导致更好的治疗和预防护理。

DESCRIPTION (provided by applicant): Project Summary/Abstract: a) We propose to develop an economical, automated system (BioSprint96) for preparing sub-megabase (250-500kb), and preferably megabase-size genomic DNA from blood or buccal swabs. b) Similar protocols on the same system will enable the targeted extraction of specific, haploid gene regions through Haplotype-Specific Extraction (HSE). This project will result in the development of new protocols for DNA sample preparation on existing instrumentation that significantly improve the length of extracted DNA compared to current methods. The proposed process is expected to be significantly faster and more cost effective than existing methods for the isolation of high molecular weight DNA and sample preparation for individual haplotyping. The completion of the project will result in a generic DNA isolation and molecular haplotype preparation technology with broad applicability in biomedical research, such as association studies, disease prognosis, pharmacogenomics, oncology testing and genomics in general. We will use specific probes and magnetic beads to separate sub-megabase and megabase diploid, genomic DNA into its haploid components based on signature sequences and polymorphic components as small as single nucleotides (SNPs). These very-long-strand, haploid genomic DNA preparations can then directly be used in conventional genotyping processes such as real-time PCR, sequencing or array-based SNP analyses to determine long range molecular haplotype data. We will develop kits for up to 3 common applications. Project Narrative: The goal of the current project of isolating large DNA fragments is to produce extended, haploid gene regions of unaltered genomic DNA. Since the method works on unamplified, genomic DNA of individual patients, it retains the DNA fragments in their original state, for instance with their epigenetic methylation pattern preserved, and does not require statistical inference to derive haplotypes. This will provide an improved tool to characterize potentially complex genetic signatures for organ transplantation (such as genes of the Major Histocompatibility Complex, MHC, and Killer Immunoglobulin-like Receptor, KIR, loci) and genetic disorders. By isolating and separating large regions of DNA into its parental fractions, unique genetic differences contributed by each parent can be used to try and link complex genetic disorders to particular allele combinations or haplotypes. This understanding should lead to a better understanding of the etiology of genetic disorders, which should ultimately result in better treatments and preventive care.