ADDRESSING THE ISSUE OF GAP REGIONS IN GENOME SEQUENCING

描述：（改编自研究者摘要）人类基因组 人类基因组计划（HGP）承诺在2010年前完成人类基因组测序 2005.申请人估计，测序工作的大约一半是 目前致力于人工序列编辑和定向测序“缺口 地区“间隙区域的出现是因为各种各样的测序假象 包括定向寡核苷酸引物的错误引发， 凝胶电泳，以及抑制DNA的稳定结构的形成 聚合酶。此外，DNA寡核苷酸杂交阵列和其他 正在开发的基因组序列验证技术， 基因诊断，也显示出高发生率的文物。这些文物是 这是由于形成了错配和模板二级结构。解决这些 问题，热力学规则管辖的DNA杂交和二级 将确定结构形成。沃森-克里克配对规则不是 足以设计稳健的DNA引物或杂交阵列。这是 因为真实的杂交反应涉及“匹配的”和“不匹配的”之间的竞争， “错配”结合位点以及靶中的分子内折叠， 探针DNA此外，杂交平衡对溶液敏感， 条件DNA折叠也改变了扩散性质，导致差的扩散。 片段的分离（例如电泳条带压缩）。一套完整 的热力学参数的沃森-克里克对，所有内部单 不匹配，经验Na+浓度依赖性已经被 测定这些新数据显著改善了杂交预测。到 进一步提高预测的质量，热力学测量将是 确定了各种常见的环图案在各种解决方案 条件这个数据库将被纳入新的算法引物 设计、碱基识别和杂交阵列的设计。新型改性 被称为“结构破坏剂”的寡核苷酸被提议用来破坏稳定的DNA 抑制DNA聚合酶的持续合成能力的二级结构， 有助于对困难的基因组缺口区域进行测序。研究者 预测，拟议中的研究将加速基因组的生产， 测序中心，降低测序成本。此外该 研究人员预测，这一信息将发现广泛的应用， 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.