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30亿个碱基对基因型。使用随机霰弹枪克隆方法，对人类大小的基因组进行测序所需的最少模板数量是6000万个，并伴有等量的相关测序反应。本课题的目标是开发一种集成的克隆和测序系统，该系统将多重模板纯化3倍，DNA测序6倍于传统方法。建立了一种新的载体，在一次连接反应中共克隆三个DNA片段作为单一模板进行分离。因此，一个三重模板相当于三个传统的质粒纯化。三个克隆片段两侧的六个引物结合位点允许从单个模板进行六组DNA测序反应。多重测序反应的简单策略使该步骤的通量比传统方法提高了6倍。三联体克隆/六联体测序系统与现有的方法和仪器完全兼容，不需要开发专门的设备来成功实施。建议的商业应用：三联体克隆/六联体测序系统将为人类基因组计划提供一种简单的方法，以减少模板纯化三次和DNA测序反应六次的数量。这种组合克隆和测序方法将大大节省时间、人力和昂贵的试剂，并提高现有方法的吞吐量。

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.