Jo Kyubong, Schramm Timothy M, Schwartz David C
Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA.
Methods Mol Biol. 2009;544:29-42. doi: 10.1007/978-1-59745-483-4_3.
Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels, creating molecular barcodes, which are efficiently read using fluorescence resonance energy transfer techniques for minimizing noise from unincorporated labels. As such, our integrative approach for the realization of genomic analysis through nanoconfinement, named nanocoding, was demonstrated through the barcoding and mapping of bacterial artificial chromosomal molecules, thereby providing the basis for a high-throughput platform competent for whole genome investigations.
单DNA分子分析方法在分析基因组科学中发挥着越来越核心的作用，因为单分子技术本质上能够对选定分子进行个体化测量，不受大量技术的限制，大量技术会盲目地对噪声进行平均并掩盖微量分析物成分的存在。因此，所有旨在进行基因组分析的单分子方法都必须应对的一个主要挑战是如何固定和操纵DNA分子，以进行有助于构建大型生物学相关数据集的测量。为应对这一挑战，本章讨论了一种用于微纳制造设备的集成方法，该方法用于在纳米尺度几何结构中操纵细长的DNA分子。理想情况下，当通道尺寸在几十纳米以内时，大的DNA线圈会通过纳米限制而伸展。重要的是，所有处理大型基因组底物的分析平台都需要跨越数百千碱基对的拉伸且通常固定的DNA分子，因为成像技术从通常以自由溶液形式存在的分子中获取序列信息，这些分子是无特征的随机线圈，类似于松散的毛线球。然而，制造尺寸足够小以促进分子拉伸的纳米级设备由于需要特殊的制造技术、昂贵的材料以及较差的操作效率而不实用。在本章中，通过讨论一种新的DNA呈现和分析方法来解决这些问题，该方法通过降低离子强度建立可扩展的纳米限制条件；使DNA分子变硬，从而能够使用易于制造且可大规模生产的设备对其进行排列以进行分析。这种新的DNA纳米限制方法辅以一种新型标记方案的开发，该方案用于用荧光染料标记可靠地标记单个分子，创建分子条形码，使用荧光共振能量转移技术可以有效地读取这些条形码，以最小化未结合标记产生的噪声。因此，我们通过纳米限制实现基因组分析的集成方法，即纳米编码，通过细菌人工染色体分子的条形码化和映射得到了证明，从而为能够进行全基因组研究的高通量平台提供了基础。
