Sorted libraries of error-free long oligonucleotides

产品说明：合成生物学充满了希望，从发现和生产新的药物，靶向治疗修改生物体，如酵母或大肠杆菌。大肠杆菌或创造全新的，更高的作物产量，二氧化碳封存到生物能源项目。然而，这些目标一直难以达到，主要是由于从头基因合成和合成新的控制和调节代谢途径的困难，一直到从化学合成的DNA合成染色体。就像任何可逆化学反应一样，通过顺序添加单体（A、C、G、T）的DNA片段合成在每一步产生小于100%的产率（典型的逐步产率为~97-99%），因此待合成的DNA的长度越长，最终纯产物的分数越小。消除错误 花费大量时间和金钱。该项目的目标是以低于当今不纯DNA片段的成本提供无错误的长寡核苷酸（100至300聚体），以释放合成生物学的潜力。我们建议建立克隆扩增的 和序列验证的长寡核苷酸。我们的长期目标是实现可负担得起的定制合成序列验证的长寡核苷酸文库的商业服务。 在I期期间，我们将1）证明单个寡核苷酸分子可以被克隆扩增和序列验证，2）证明携带克隆扩增的珠可以被捕获在携带序列特异性探针的微阵列上，以及3）证明序列验证的寡核苷酸能够以十倍降低的错误率进行基因组装。 在第II阶段，我们将1）开发一种方案/装置，以标准化从文库中调查的每种寡核苷酸的数量，2）建立用于生产经序列验证的寡核苷酸的大型文库的标准操作程序，以及3）确定可作为商业产品提供的最大寡核苷酸长度。 公共卫生相关性：序列验证长寡核苷酸的可负担得起的定制文库的前所未有的可用性将使更快，更容易和更便宜的基因和大DNA片段组装在合成生物学中具有重要应用。这项技术无疑将有助于发现新的细胞机制、分子治疗工具甚至药物，最终造福社会。

DESCRIPTION: Synthetic Biology is full of promises ranging from discovery and production of new drugs, targeted therapies modifying organisms such as yeast or E. coli or creating totally new ones, higher crop yields, CO2 sequestration to bio-energy projects. However, these goals have been difficult to reach primarily due to the difficulty of De Novo gene synthesis and synthesis of new control and regulatory metabolic pathways all the way to synthetic chromosomes from chemically synthesized DNA. Just like any reversible chemical reaction DNA fragment synthesis by sequentially adding the monomers (A,C,G,T) gives less than 100% yield at each step( typical stepwise yields are ~97-99%) as a result the longer the length of DNA to be synthesized the smaller is the fraction of pure product at the end. Elimination of the errors take significant time and money. The goal of this project is to provide error free long oligonucleotides (100 to 300 mers) at a cost lower than the impure DNA fragments of today to unlock the potential of synthetic biology. We are proposing to make libraries of clonally amplified and sequence verified long oligonucleotides. Our long term objectives are to implement a commercial service of affordable custom synthesis of sequence-verified long oligonucleotide libraries. During Phase I, we will 1) to demonstrate that single oligonucleotide molecules can be clonally amplified and sequence verified, 2) to demonstrate that beads bearing clonal amplifications can be captured on a microarray bearing sequence-specific probes and 3) to demonstrate that sequence verified oligonucleotides enable gene assembly with ten fold reduced error rate. During Phase II, we will 1) to develop a protocol/device to normalize the number of each oligonucleotide surveyed from a library, 2) to establish a standard operating procedure for the production of large libraries of sequence-verified oligonucleotides and 3) to determine the maximum oligonucleotide length that could be offered as a commercial product. PUBLIC HEALTH RELEVANCE: The unprecedented availability of affordable custom libraries of sequence verified long oligonucleotides will enable faster, easier and cheaper gene and large DNA fragment assembly with major applications in Synthetic Biology. This technology will undoubtedly bolster the discovery of new cellular mechanisms, molecular therapeutic tools, or even drugs, ultimately benefiting the society.