High Fidelity Linear MicroVector to Clone Complex, Problematic, and Large DNAs

这项研究的目标是显著提高克隆和分析大型和不稳定的能力 DNA片段。我们建议开发一种新型的线性克隆载体来最大限度地提高克隆的稳定性 细菌宿主中的DNA。该载体将使用噬菌体Phi29的复制蛋白来实现 所有DNA插入的复制准确率最高，包括富含AT、重复或结构不稳定的DNA插入 基因。对于任何大小范围(0-100kb)的DNA，克隆方法将是快速和容易的，只需要 将小载体臂连接到DNA上，然后转化细菌。这个系统将会 超过现有的稳定性、保真度和无偏见标准，超过了我们以前 构建了pJAZZ线性克隆载体。重要的是，它将允许直接克隆Fosmide-或 BAC大小的DNA。 该载体将具有最小的细菌序列，为哺乳动物细胞提供显著的好处 转基因、治疗性蛋白质生产、干细胞研究，以及最终的基因治疗。这个 克隆的DNA将与核定位信号结合，以增强在 哺乳动物细胞。 迫切需要这种Phi29载体来帮助发现人类疾病的治疗方法。例如， 人类疟疾寄生虫恶性疟原虫的基因组是最难识别的基因组之一。 克隆，由于其重复性和高度富含AT的性质。我们将与惠康信托桑格合作 恶性疟原虫长插入基因组文库的构建及测序。这个独一无二的 为了能够对这种致命病原体进行基因研究，迫切需要资源。

The goal of this research is to dramatically improve the ability to clone and analyze large and unstable DNA fragments. We propose to develop a novel linear cloning vector to maximize stability of cloned DNA in the bacterial host. The vector will use the replication proteins of the phage Phi29 to achieve the highest accuracy of replication for all DNA inserts, including AT-rich, repetitive, or structurally unstable genes. The cloning method will be quick and easy for DNA of any size range (0-100 kb), only requiring ligation of small vector arms to the DNA, followed by transformation of bacteria. This system will surpass existing standards for stability, fidelity, and lack of bias, exceeding the limits of our previously developed pJAZZ linear cloning vector. Importantly, it will enable straightforward cloning of fosmid- or BAC-sized DNAs. The vector will have minimal bacterial sequences, providing significant benefits for mammalian cell transfection, therapeutic protein production, stem cell research, and ultimately gene therapy. The cloned DNAs will be bound to nuclear localization signals for enhanced delivery and expression in mammalian cells. This Phi29 vector is urgently needed to help discover treatments for human illness. For example, the genome of the human malaria parasite, Plasmodium falciparum, is among the most difficult genomes to clone, due to its repetitive, highly AT-rich nature. We will collaborate with the Wellcome Trust Sanger Institute to construct and sequence long-insert genomic libraries of Plasmodium falciparum. This unique resource is critically needed to enable genetic research on this deadly pathogen.