Temperature Gradient Hybridizer and Real-time Imager for DNA Microarrays

描述(申请人提供)：由于可以快速获取大量的遗传信息，DNA微阵列已成为生物学研究中不可或缺的工具。然而，尽管它们有用，但一些实验困难导致同一实验室之间甚至同一实验室内的数据质量差异很大。更困难的挑战之一是，相同长度的DNA探针可能具有差异很大的杂交或熔化温度(TM‘s)。令人惊讶的是，尽管存在用于快速优化反应条件的温度梯度聚合酶链式反应装置，但没有用于微阵列的商业装置。其结果是，目前分子生物学中一些最昂贵的实验使用了次优条件。 该项目致力于开发一种硬件系统，用于为任意微阵列设计中包含的每个探头产生最佳的“温度地址”。这一改进将极大地提高核酸微阵列数据的准确性和重复性。通过提高结合的特异性，也应该提高对样本中稀有序列的检测限。最后，该系统将极大地提高灵活性，使其能够包括以前将从探针集中丢弃的序列。随着微阵列在生物研究中不断扩大的应用，所开发的系统将代表着该领域的重要进步，应该具有相当大的商业吸引力。在第二阶段，我们将把温度梯度杂交硬件与基于CCD的成像系统集成在一起，以实现实时图像采集。实时成像将允许遵循动力学以及非等温(在这种情况下为时变梯度)数据的获取，例如，退火动力学、荧光熔化曲线、竞争结合分析和芯片上的聚合酶链式反应。到第二阶段结束时，我们将能够向微阵列成像和支持设备的制造商销售我们特性良好的设备以及原理证明数据。 DNA微阵列已经成为生物学研究不可或缺的工具，但一些技术挑战阻碍了它们的可靠使用。该项目的重点是开发设备，为微阵列上的每个探针提供最佳杂交条件，以及实时成像阵列上的表面反应。由此产生的工程进步将使微阵列设计在各种基因分型应用中具有更大的灵活性，包括基因表达分析、染色体可获得性、杂交测序和微生物鉴定。

DESCRIPTION (provided by applicant): Because of the large amount of genetic information which can be rapidly accessed, DNA microarrays have become indispensable tools for biological investigations. Despite their utility, however, a number of experimental difficulties have resulted in widely varying data quality between and even within the same laboratories. One of the more difficult challenges is that DNA probes of the same length can have widely varying hybridization or melting temperatures (Tm's). Surprisingly, although temperature gradient PCR devices exist for rapid optimization of reaction conditions, no such commercial devices exist for microarrays. The result is that suboptimal conditions are used for what are presently some of the most expensive experiments in molecular biology. This project focuses on the development of a hardware system for producing optimal "temperature addresses" for every probe included in an arbitrary microarray design. This improvement will dramatically improve the accuracy and reproducibility of nucleic acid microarray data. By improving specificity of binding, the limit of detection for rare sequences in a sample should also be improved. Finally, the system will greatly increase flexibility regarding the ability to include sequences which would have been previously discarded from probe sets. As microarrays continue to find expanding applications in biological research, the developed system will represent an important advance in the field and should have considerable commercial attractiveness. In Phase II, we will integrate the temperature gradient hybridization hardware with a CCD-based imaging system for real-time image acquisition. Real-time imaging will allow kinetics to be followed as well as non-isothermal (time-varying gradients in this case) data to be acquired such as annealing kinetics, fluorescence melting curves, competitive binding assays, and "on-chip" PCR, for example. By the end of Phase II, we will able to market our well-characterized equipment as well as the proof- of-principle data to manufacturers of microarray imaging and support equipment. DNA microarrays have become an indispensable tool for biological research, however a number of technical challenges have hampered their reliable use. This project focuses on developing the equipment for providing the optimal hybridization conditions for every probe on a microarray, as well as for imaging the surface reactions on the array in real-time. The engineering advances which will result will allow increased flexibility in microarray design for a variety of genotyping applications including gene expression analysis, chromosomal accessibility, sequencing by hybridization, and microbial identification.