ADDRESSING THE ISSUE OF GAP REGIONS IN GENOME SEQUENCING

描述：(改编自研究人员摘要)人类基因组 人类基因组计划(HGP)承诺在今年之前提供完整的人类基因组序列 2005年。申请人估计，大约一半的测序工作是 目前致力于手动序列编辑和定向测序 差距区域的出现是因为各种测序伪像的出现 包括定向寡核苷酸引物的错误引发，条带压缩 凝胶电泳，以及抑制DNA的稳定结构的形成 聚合酶。此外，DNA寡核苷酸杂交阵列等 正在开发的用于基因组序列验证和 基因诊断显示，伪影的发生率也很高。这些文物是 由于不匹配和模板二级结构的形成。要解决这些问题 问题、DNA杂交和二次杂交的热力学规则 将确定结构的形成。沃森-克里克配对规则不是 足以设计强大的DNA引物或杂交阵列。这是 因为真正的杂交反应涉及“匹配”和 靶标和靶分子中的“错配”结合部位和分子内折叠 探测DNA。此外，杂交平衡对溶液很敏感。 条件。DNA折叠也会改变扩散特性，导致不良反应 片段分离(例如，电泳带压缩)。全套设备 沃森-克里克对的热力学参数，全部为内部单项 不匹配，并且经验上的Na+浓度依赖关系已经 下定决心。这些新数据显著改善了杂交预测。至 进一步提高预报质量，将热力学测量 针对各种解决方案中常见的各种环路主题进行确定 条件。这个数据库将被合并到新的Primer算法中 杂交阵列的设计、碱基调用和设计。新颖的改装 被称为“结构破碎物”的寡核苷酸被提出用来破坏稳定的DNA。 抑制DNA聚合酶的加工能力的二级结构 便于对困难的基因组缺口区域进行测序。调查员 预测拟议中的研究将加速基因组的产生 测序中心，降低测序成本。此外， 研究人员预测，这些信息将在以下领域得到广泛应用 生物技术和生物信息学。

DESCRIPTION: (Adapted from the investigator's abstract) The Human Genome Project (HGP) promises to deliver a complete human genome sequence by the year 2005. The applicant estimates that about one half of the sequencing effort is devoted currently to manual sequence editing and directed sequencing of "gap regions." Gap regions occur because of a variety of sequencing artifacts including mispriming of directed oligonucleotide primers, band-compressions in gel electrophoresis, and the formation of stable structures that inhibit DNA polymerase. In addition, DNA oligonucleotide hybridization arrays and other technologies, which are being developed for genome sequence verification and gene diagnostics, also show a high incidence of artifacts. These artifacts are due to formation of mismatched and template secondary structure. To solve these problems, the thermodynamic rules governing DNA hybridization and secondary structure formation will be determined. The Watson-Crick pairing rules are not sufficient to design robust DNA primers or hybridization arrays. This is because real hybridization reactions involve competition among "matched" and "mismatched" binding sites as well as intramolecular folding in the target and probe DNAs. In addition, hybridization equilibria are sensitive to solution conditions. DNA folding also alters diffusion properties, resulting in poor separation of fragments (e.g. electrophoresis band compression). A complete set of thermodynamic parameters for Watson-Crick pairs, all internal single mismatches, and an empirical Na+ concentration dependence has already been determined. These new data significantly improve hybridization predictions. To further improve the quality of predictions, thermodynamic measurements will be determined for various commonly occurring loop motifs in a variety of solution conditions. This database will be incorporated into new algorithms for primer design, base calling, and design of hybridization arrays. Novel modified oligonucleotides, called "structure breakers," are proposed to break stable DNA secondary structures that inhibit the processivity of DNA polymerases and to facilitate sequencing of difficult genome gap regions. The investigator predicts that the proposed studies will accelerate the production of genome sequencing centers and reduce the cost of sequencing. In addition, the investigator predicts that this information will find wide applications in biotechnology and bioinformatics.