SEQUENCING WITH SHORT OLIGONUCLEOTIDES

使用短寡核苷酸测序

• 批准号: 2209452
• 负责人: Susan H Hardin
• 金额: $ 10.38万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2209452
• 关键词: biomedical automation; biotechnology; gel electrophoresis; genetic library; genetic techniques; nucleic acid sequence; oligonucleotides; robotics

In order to meet the goals of the Human Genome Program, literally billions of bases of DNA must be sequenced. To accomplish this feat, technology must advance so that the amount of bases determined is vastly increased, the quality of the data is highly accurate and the cost per base is significantly decreased, ie faster, better, cheaper. Additionally, it would be advantageous if technological advances, in addition to impacting large sequencing projects, would benefit researchers in laboratory settings where researchers have access to cost effective equipment that serve a multitude of purposes. Two types of sequencing technologies are being developed and are related by their use of short oligonucleotides. One technology, Cyclic Ligation Sequencing (CLS), uses a thermal cycling procedure to anneal hexamers to a dsDNA template, ligate the hexamers using T4 DNA ligase, denature the ligated primers from the template, and repeatedly cycle this temperature regime. Subsequently, the ligated hexamers are used to prime a DNA sequencing reaction. Optimization of CLS is dependent upon optimization of the ligation reaction. Another technology uses octamers to efficiently prime a DNA sequencing reaction. Optimization of octamer sequencing depends on experimentally determining the rules important in designing octamers which produce high quality sequence data and constitute a reasonably sized primer library. Both CLS and octamer sequencing would be faster; the existence of a primer library eliminates the delay caused waiting for the next primer to be synthesized. Both yield sequencing results equivalent to or better than traditional primer walking due to pre-selection of optimal primers. Both technologies would be significantly cheaper; standard sequencing primers, the major cost in the reaction, would be replaced by a library composed of short oligonucleotides able to prime multiple reactions.

为了实现人类基因组计划的目标， DNA的碱基必须被测序。为了实现这一壮举，技术 必须推进，以使确定的碱基数量大大增加， 数据的质量非常准确，每个基地的成本 大大降低了（更快、更好、更便宜）。而且 如果技术进步，除了影响 大型测序项目，将有利于实验室研究人员 研究人员可以使用具有成本效益的设备， 服务于多种目的。两种类型的测序技术是 正在开发中，并通过使用短寡核苷酸而相互关联。 一种技术，环状连接测序（CLS），使用热循环 将六聚体退火至dsDNA模板的程序， 使用T4 DNA连接酶，使连接的引物从模板变性，和 反复循环该温度状态。随后， 六聚体用于引发DNA测序反应。 CLS的优化 依赖于连接反应的优化。另一 技术使用八聚体来有效地引发DNA测序反应。 八聚体测序的优化取决于实验确定 设计高质量八聚体的重要规则 序列数据并构成合理大小的引物文库。两个CLS 八聚体测序会更快;引物库的存在 消除了等待下一个引物合成所引起的延迟。 这两种方法的测序结果都相当于或优于传统的 由于预先选择了最佳引物，因此进行了引物步移。 这两种技术 会便宜得多;标准测序引物，主要是 在反应成本，将被取代的图书馆组成的短 寡核苷酸能够引发多个反应。