2-D Microfluidic Gene Scanner

二维(2-D)DNA凝胶电泳法可以根据DNA片段的大小和序列的不同在两个维度上进行分辨。除了核苷酸测序，这项技术提供了唯一一种理论上灵敏度几乎为100%的突变检测方法。目前，与其他突变检测技术相比，二维DNA凝胶电泳法具有较高的性价比，但不是大规模DNA分析的高通量平台。尽管传统的二维DNA分析具有选择性和灵敏度，但目前使用的这项技术是一项人工密集型和耗时任务的集合，容易出现重复性和定量准确性差的问题。为了提高产量和降低成本，自动化和小型化是有益的策略。我们提出的研究的具体目标是(I)开发一种在一次性塑料基质上使用微流控网络的二维微流控基因扫描仪，以及(Ii)在第一阶段项目中以大肠杆菌和酵母基因组为模型系统来演示二维微流控基因扫描仪的速度和分辨率。在展示了用于快速和高分辨率DNA分析的二维微流控基因扫描仪后，我们将把这项技术与计算机设计的聚合酶链式反应引物结合起来，在一次反应中产生大量DGGE优化的目标片段，即聚合酶链式反应多重反应。我们的目标是开发并商业化一种广泛的并行基因扫描仪，用于检测大型人类疾病基因的突变，并在基于人群的研究中探索人类遗传变异性。拟议的商业应用：拟议研究中将开发的技术是大规模并行、高通量基因组、筛查和快速彻底评估基因突变的新工具。2-D基因扫描仪的潜在客户很广泛，包括对高速诊断、疾病研究、基因治疗开发和药物发现感兴趣的医疗诊断和生物制药公司。

Two-dimensional (2-D) DNA gel electrophores allows DNA fragments to be resolved in two dimensions based on their differences in size and sequence. Apart from nucleotide sequencing, this technique provides the only method with a theoretical sensitivity of virtually 100% for mutation detection. At present, 2-D DNA gel electrophoresis is relatively cost-effective in comparison with other mutation detection techniques, but it is not a high-throughput platform for large-scale DNA analysis. Despite the selectivity and sensitivity of conventional 2-D DNA analysis, this technique as practiced today is a collection of manually intensive and time-consuming tasks, prone to poor reproducibility and quantitative accuracy. To increase throughput and decrease cost, automation and miniaturization are beneficial strategies. The specific aims of our proposed research are to (i) develop a 2-D microfluidic gene scanner employing microfluidic networks in disposable plastic substrates, and (ii) demonstrate the speed and resolving power of the 2-D microfluidic gene scanner using the E. coli and yeast genomes as model systems during the Phase I project. Upon demonstration of the 2-D microfluidic gene scanner for rapid and high resolution DNA analysis, we will integrate the technology with the computerized design of PCR primers which generate a large number of DGGE-optimized target fragments in one single reaction, i.e. a PCR multiplex. Our goal is to develop and commercialize an extensive parallel gene scanner for mutation detection in large human disease genes and exploring human genetic variability in population-based studies. PROPOSED COMMERCIAL APPLICATIONS: The technology to be developed in the proposed research is a new tool for massively parallel, high throughput genome, screening, and rapid and thorough evaluation of genetic mutation. Potential customers for the 2-D gene scanner instrument are widespread, including medical diagnostics and biopharmaceutical companies interested in high speed diagnostics, disease research, gene therapy development, and drug discovery.