Hybridization Chain Reaction: In Situ Amplification for Biological Imaging

杂交链式反应：生物成像的原位放大

• 批准号: 7125451
• 负责人: NILES A PIERCE
• 金额: $ 22.22万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-22 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7125451
• 关键词: bioimaging /biomedical imaging; chick embryo; fluorescence microscopy; fluorimetry; gene expression; genetic markers; genetic regulation; genetic regulatory element; immunocytochemistry; in situ hybridization; nanotechnology; neurogenetics; nucleic acid amplification techniques; oligonucleotides; rhombencephalon; technology /technique development; thermodynamics

DESCRIPTION (provided by applicant): The recent explosion of genomic data has offered an unprecedented opportunity to study the molecular basis for developmental and disease processes. Although microarray techniques for profiling the genes expressed in a given tissue have become commonplace, methods for determining the exact spatial and temporal extent of gene expression are still in need of improvement. In situ hybridization techniques for gene expression studies often have limited sensitivity and significant background signal even in locations where no target genes are present. Furthermore, existing technologies do not fully exploit the potential for imaging many genes simultaneously. The goal of the proposed research is to adapt a newly developed nanosensor technology for multiplexed in situ amplification of gene expression. This amplification tool, termed hybridization chain reaction (HCR), reduces background by activating only when a probe molecule binds specifically to its target. This event triggers the self-assembly of a tethered 'polymer' from fluorescently-labeled DMA hairpins. Parallel multiplexing can be achieved simply by using independent HCR amplifiers for each unique target species. The research plan involves the design, validation and application of in situ HCR amplifiers. Specific aims are: 1: Design triggered, multiplexed, nonlinear HCR amplifiers, and refine the computational tools for the design of HCR amplifiers. 2: Validate the spatial localization, sensitivity, specificity and multiplexing of the amplifiers in situ using nanolithography techniques to precisely pattern target molecules on surfaces. 3: Apply HCR to in situ hybridization of patterning genes in avian embryos, testing for the co- expression of genetic markers predicted by previous studies. The objective is to develop in situ HCR amplifiers that will enable the sensitive and simultaneous detection of numerous targets in biological specimens. If successful, this nanotechnology will serve as an important adjunct to modern genomic and proteomic tools in settings ranging from biological experiments to tissue biopsies.

描述(由申请人提供)：最近基因组数据的爆炸性增长为研究发育和疾病过程的分子基础提供了前所未有的机会。尽管基因芯片技术在特定组织中表达的基因图谱已经变得很常见，但确定基因表达的确切空间和时间范围的方法仍然需要改进。用于基因表达研究的原位杂交技术通常具有有限的灵敏度和显著的背景信号，即使在没有靶基因的位置也是如此。此外，现有技术没有充分开发同时对多个基因进行成像的潜力。 这项拟议的研究的目标是采用一种新开发的纳米传感器技术来进行基因表达的多路原位扩增。这种被称为杂交链式反应(HCR)的扩增工具，仅在探针分子与其靶标特异结合时才激活，从而减少了背景。这一事件触发了来自荧光标记的DMA发夹的拴系聚合物的自组装。对于每个独特的目标物种，只需使用独立的HCR放大器即可实现并行多路传输。 该研究计划涉及原位HCR放大器的设计、验证和应用。具体目标是： 1：设计触发、多路、非线性HCR放大器，并改进用于设计HCR放大器的计算工具。 2：使用纳米光刻技术在表面精确构图目标分子，验证原位放大器的空间定位、灵敏度、特异性和多路复用性。 3：将HCR应用于禽类胚胎花纹基因的原位杂交，检测先前研究预测的遗传标记的共表达。 其目的是开发原位HCR放大器，使其能够灵敏地同时检测生物标本中的许多目标。如果成功，这项纳米技术将成为现代基因组和蛋白质组工具的重要辅助工具，适用于从生物实验到组织活检的各种环境。