SEQUENCING WITH SHORT OLIGONUCLEOTIDES

使用短寡核苷酸测序

基本信息

批准号：
2674221
负责人：
Susan H Hardin
金额：
$ 9.69万
依托单位：
UNIVERSITY OF HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-09-30 至 2000-08-31
项目状态：
已结题

项目摘要

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.

为了满足人类基因组计划的目标，实际上是数十亿必须对DNA的碱基进行测序。为了实现这一壮举，技术必须前进，以使确定的基础数量大大增加，数据的质量高度准确，每个基础成本为显着下降，即更快，更好，更便宜。另外，它如果技术进步，除了影响技术方面，将是有利的大型测序项目将使研究人员受益于实验室研究人员可以使用具有成本效益的设备的设置提供多种用途。两种类型的测序技术是通过使用短寡核苷酸来开发并与它们相关。一项技术，即循环连接测序（CLS），使用热循环将六聚体退火为dsdna模板的程序，将六聚体连接使用T4 DNA连接酶，使模板的连接底漆变性，然后反复循环这种温度状态。随后，连接六聚体用于给DNA测序反应产生。 CLS的优化取决于连接反应的优化。其他技术使用八聚体有效启动DNA测序反应。八聚体测序的优化取决于实验确定这些规则对于设计产生高质量的八聚体很重要序列数据并构成一个合理尺寸的引物库。两个CL 八聚体测序将更快；底漆库的存在消除导致等待下一个底漆的延迟。两者的屈服测序结果等于或比传统更好底漆步行是由于最佳底漆的预选而导致的。这两种技术会更便宜；标准测序引物，主要反应中的成本将由由简短组成的库代替寡核苷酸能够产生多种反应。