A Single-Molecule Barcoding System using Nanoslits for DNA Analysis: Nanocoding
A Single-Molecule Barcoding System using Nanoslits for DNA Analysis: Nanocoding
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DOI：
10.1007/978-1-59745-483-4_3
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发表时间：
2009-01-01
期刊：
影响因子：
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通讯作者：
Schwartz, David C.
中科院分区：
文献类型：
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作者：
Jo, Kyubong;Schramm, Timothy M.;Schwartz, David C.
单DNA分子方法在分析基因组科学中发挥着越来越重要的作用，因为单分子技术本质上提供了所选分子的个性化测量，不受批量技术的限制，批量技术盲目地平均噪声并掩盖次要分析物组分的存在。因此，针对基因组分析的所有单分子方法必须解决的主要挑战是如何扩增和操纵DNA分子以用于促进构建大型生物相关数据集的测量。为了迎接这一挑战，本章讨论了一个集成的方法，用于操纵细长的DNA分子在纳米几何形状的微制造和纳米制造设备。理想情况下，当通道尺寸在几十纳米内时，大的DNA螺旋通过纳米限制拉伸。重要的是，所有使用大型基因组底物的分析平台都需要跨越数百个内切酶对的拉伸的、通常固定的DNA分子，因为成像技术从通常存在于自由溶液中的分子中获取序列信息，这些分子作为类似于松软纱线球的未显示的随机卷曲。然而，由于需要特殊的制造技术、昂贵的材料和差的操作效率，以足够小的尺寸制造的纳米级器件促进分子拉伸使得这些器件不切实际。在这一章中，这样的问题是通过讨论一种新的方法来解决DNA的介绍和分析，建立可缩放的纳米约束条件，通过减少离子强度，硬化DNA分子，从而使其阵列分析使用容易制造的设备，也可以大规模生产。这种新的DNA纳米限制方法通过开发一种新的标记方案来补充，该方案用于可靠地标记单个分子，使用荧光染料标记，创建分子条形码，使用荧光共振能量转移技术有效地读取，以最大限度地减少未掺入标记的噪声。因此，我们通过纳米限制实现基因组分析的综合方法，称为纳米编码，通过细菌人工染色体分子的条形码和映射得到了证明，从而为高通量平台提供了基础，适合全基因组研究。
Single DNA molecule approaches arc 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 arc within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs arc 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 arc 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.