SEQUENCING WITH SHORT OLIGONUCLEOTIDES

为了实现人类基因组计划的目标，字面意思是数十亿 必须对DNA碱基进行测序。为了完成这一壮举，技术 必须向前推进，以使所确定的碱基量大大增加， 数据的质量非常准确，每个基数的成本是 明显减少了(更快、更好、更便宜).此外，它还 将是有利的，如果技术进步，除了影响 大型测序项目将使实验室的研究人员受益 研究人员可以使用具有成本效益的设备的环境 服务于多种目的。有两种测序技术 正在开发中，并通过使用短寡核苷酸而相关。 一种名为环连接测序(CLS)的技术使用了热循环 将六聚体退火至dsDNA模板，连接六聚体的程序 使用T4 DNA连接酶，从模板中变性连接的引物，以及 反复循环这一温度制度。随后，结扎的 六聚体被用来启动DNA测序反应。CLS的优化 依赖于连接反应的优化。另一个 技术使用八聚体来有效地启动DNA测序反应。 八聚体测序的优化依赖于实验确定 在设计生产高质量的八聚体时的重要规则 测序数据，并构建一个大小合理的引物库。两个CLS 而八聚体测序会更快；如果有一个引物库 消除了等待下一次底漆合成所造成的延迟。 两者的测序结果都与传统测序结果相同或更好 由于预选了最佳的引发剂，所以引发剂的走动。这两种技术 会便宜得多；标准测序引物，主要是 在反应中，成本将被一个由短期组成的图书馆所取代 能够引发多种反应的寡核苷酸。

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.