AN INTEGRATED MULTIPLEX CLONING AND SEQUENCING SYSTEM

人类基因组计划的一个目标是确定智人的30亿碱基对基因型。使用随机鸟枪克隆方法，测序人类大小的基因组所需的最小模板数量为6000万，具有相等数量的相关测序反应。本提案的目标是开发一种集成的克隆和测序系统，该系统将比常规方法多路复用模板纯化三倍和DNA测序六倍。开发了一种新的载体，用于在单个连接反应中共克隆三个DNA片段，以作为单个模板分离。因此，一个三链体模板相当于三个常规质粒纯化。侧接三个克隆片段中的每一个的六个引物结合位点允许来自单个模板的六组DNA测序反应。多重测序反应的简单策略将该步骤的通量提高了常规方法的六倍。三链体克隆/六链体测序系统与现有的方法和仪器完全兼容，并且不需要开发用于成功实施的专门仪器。拟定商业应用：三链克隆/六链测序系统将为人类基因组计划提供一种简单的方法，用于减少模板纯化的次数和DNA测序反应的次数。这种组合克隆和测序方法将导致在时间、劳动力和昂贵试剂方面的显著节省，以及提高现有方法的通量。

One goal of the Human Genome Initiative is to determine the 3 billion base pair genotype of Homo sapiens. The minimum number of templates required to sequence a human sized genome is 60 million using random shotgun cloning methods, with an equivalent number of associated sequencing reactions. The goal of this proposal is to develop an integrated cloning and sequencing system which will multiplex template purification three fold and DNA sequencing six fold over conventional methods. A new vector was developed to co-clone three DNA fragments in a single ligation reaction for isolation as a single template. Thus, one triplex template is equivalent to three conventional plasmid purifications. The six primer binding sites flanking each of the three cloned fragments permits six sets of DNA sequencing reactions from a single template. A simple strategy to multiplex the sequencing reactions improves the throughput of this step by six fold over conventional methods. The triplex cloning/sextaplex sequencing system is completely compatible with existing methods and instrumentation and does not require the development of specialized apparatus for successful implementation. PROPOSED COMMERCIAL APPLICATION: The triplex cloning/sextaplex sequencing system will provide the Human Genome Project with a simple method for reducing the number of template purifications three-old and DNA sequencing reactions six-old. This combinatorial cloning and sequencing method will result in significant savings in time, labor and expensive reagents, as well as improving the throughput of existing methods.