SNP Analysis by Surface Invasive Cleavage on DNA Arrays

描述(由申请人提供)：在这份竞争性续签提案中，我们寻求继续开发用于高通量SNP分析的“表面入侵者”DNA阵列。在之前的资助期间，我们开发了表面入侵者分析，并展示了直接在斑点DNA阵列上从未扩增的人类基因组DNA样本中并行分析SNPs的“原则证明”。现在我们希望将这项技术带入下一步，即在高密度DNA阵列格式中实现这种化学，其中DNA阵列是通过光刻方法制造的。为了能够制造所需的DNA阵列，需要解决三个问题。首先，将采用化学方法在5‘-gt；3’方向进行DNA分子的光刻合成。第二，将开发和实施化学，以允许在单个阵列元件中混合的两个不同序列的光刻合成；两者都是在表面形成/必需的三元络合物所必需的。第三，将开发用于光刻DNA合成的玻碳和/或金刚石薄膜基板。这项提议的第二个推力将是研究一种基于对表面单分子裂解事件的检测的方法。滚环扩增反应可以使DNA分子从一个起始点长达数十万个碱基。由于很容易检测到如此长的DNA分子，这就增加了直接检测表面单个切割的DNA分子的可能性；事实上，我们的初步结果已经显示了这种能力。这是耐人寻味的，因为它开启了分析非常低水平的核酸靶标的可能性，可能只有单个分子，在基因表达分析、传染病诊断、生物制剂检测以及从口腔拭子或指刺血样等微量样本中进行基因分型等领域有许多应用。

DESCRIPTION (provided by applicant): In this competitive renewal proposal we seek to continue the development of "surface invader" DNA arrays for high throughput SNP analysis. During the prior grant period we developed the surface invader assay and demonstrated "proof-of-principle" for the parallel analysis of SNPs directly from unamplified human genomic DNA samples on spotted DNA arrays. Now we desire to take this technology to the next step, i.e. to implement this chemistry in a high-density DNA array format, where the DNA array is manufactured by photolithographic methods. There are three issues that will be addressed to enable fabrication of the needed DNA arrays. First, chemistry will be implemented for the photolithographic synthesis of DNA molecules in the 5'->3' direction. Second, chemistry will be developed and implemented to allow the photolithographic synthesis of two different sequences intermixed in a single array element; both are needed to form the / necessary ternary complex on the surface. Third, glassy carbon and/or diamond thin film substrates will be developed for use in photolithographic DNA synthesis. A secondary thrust of this proposal will be to investigate an approach to detection based upon the detection of single molecule cleavage events on the surface. The rolling circle amplification reaction can make DNA molecules hundreds of thousands of bases long from a single initiation site. As it is very easy to detect such long DNA molecules, this raises the possibility of directly detecting individual cleaved DNA molecules on the surface; in fact, our preliminary results have shown this capability. This is intriguing, as it opens the possibility of analyzing very low levels of nucleic acid targets, perhaps as little as a single molecule, with many applications in areas such as gene expression analysis, infectious disease diagnostics, biowarfare agent detection, and genotyping from minute samples such as buccal swabs or fingerprick blood samples.