Nanopore detection of haplotype

描述（申请人提供）：我将开发使用纳米孔进行高速SNP检测和单倍型所需的技术和基础科学。该技术依赖于驱动单个多核苷酸分子通过纳米孔，与敏感的单通道记录电子设备相连，可以直接读出聚合物的特性。这些新的分子诊断方法的发展利用了三个最近的发现：(1)膜通道，或纳米孔，可以作为一种高通量设备来检测和探测单个分子，因为它们通过纳米孔转运；(2)通过纳米孔的离子电流对易位分子横截面积的局部变化敏感；(3)一种新的平面制造方法-离子束雕刻-使我们能够在坚固的固态绝缘膜上创建所需尺寸的个位数纳米级孔。我们的工作将优化：(a)锌指蛋白DNA标记；(b) DNA长度的电读数；(c)实现可靠的SNP鉴定和高速单倍型所需的锌指蛋白的最小片段长度和数量。提出的技术将影响基础研究领域，如必须处理复杂的基因和突变集的发展和癌症，以及现有的方法来快速检查大量个体的多染色体上的大量多态性位点之间的联系是有限的。提出的工具和基础研究将为未来发展高灵敏度、信息丰富的诊断方法开辟新的可能性，这些方法对早期疾病检测至关重要。

DESCRIPTION (provided by applicant): I will develop the technology and basic science needed to use nanopores for high speed SNP detection and haplotyping. The technology depends on driving single polynucleotide molecules through a nanopore coupled to sensitive single channel recording electronics that can provide a direct read-out of the polymer's characteristics. The development of these new molecular diagnostic methods takes advantage of three recent discoveries: (1) A membrane channel, or nanopore, can be used as a high-throughput device that detects and probes single molecules as they translocate through the nanopore; (2) The ionic current through the nanopore is sensitive to local changes in the cross-sectional area of the translocating molecule; (3) A new, planar fabrication method - ion beam sculpting - that allows us to create a single digit nanoscale pore of a desired dimension in robust solid state insulating membranes. Our work will optimize: (a) zinc-finger protein labeling of DNA; (b) the electrical readout of DNA length; (c) the minimum fragment length and number of different zinc-finger proteins needed to achieve reliable SNP identification and high-speed haplotyping. The proposed technologies will impact basic research areas such as development and cancer that must deal with complex sets of genes and mutations, and where existing methods to rapidly examine the linkages between a large number of polymorphic sites on multiple chromosomes in a large number of individuals are limiting. The tools and basic research proposed will open new possibilities for the future development of high sensitivity, information rich diagnostic methods that are critical for early disease detection.