Improved Molecular Barcodes by Lifetime Discrimination

描述（由申请人提供）：DNA微阵列通过其同时测量数千个基因的mRNA水平的能力推进了我们对细胞生物学的理解。然而，这些微阵列生产昂贵，并且由于将微观液滴可靠地递送到载玻片表面所涉及的技术问题，质量控制是一个问题。此外，由于混合限制，载玻片上结合反应的动力学缓慢。为了克服DNA微阵列的这些局限性，已经提出用大量的微小塑料珠进行这些测定，每个塑料珠在流式细胞仪中用独特的分子条形码识别。目前的条形码策略是基于用具有不同波长和不同加载强度的荧光染料浸渍塑料珠。理论上，使用4种不同的染料和10种不同的强度将产生10，000个条形码。然而，相邻染料的发射波长不能太接近，否则来自发射肩部的光将干扰准确的强度测量。在实践中，Luminox，Inc.提供了一组100个珠子，在两个波长下具有十个加载强度。RMD提出使用类似的概念，但基于荧光寿命而不是强度的差异的分子条形码。基于荧光寿命的区分对来自相邻染料的干扰信号几乎不敏感。辐射衰变的主要成分可以很容易地从次要的干扰成分中去卷积。这将允许使用发射最大值接近的染料，从而产生更高数量的条形码。此外，由于我们的方法不受绝对强度测量固有误差的影响，因此需要较少的校准，并且每个波长处可能有超过10个变化是可区分的。如果开发出来，这种方法将在疾病研究中得到应用。例如，可以研究大群体样本对与遗传变异相关的治疗性治疗的反应。

DESCRIPTION (provided by applicant): DNA microarrays have advanced our understanding of cellular biology by their ability to simultaneously measure mRNA levels for thousands of genes. However, these microarrays are expensive to produce and quality control is a problem due to technical issues involved in reliably delivering microscopic droplets to the surface of the slide. Also, the kinetics of the binding reaction on the glass slides is slow do due mixing limitations. In order to overcome these limitations of DNA microarrays, it has been proposed to perform these assays with a large collection of tiny plastic beads, each of which is identified with a unique molecular barcode in a flow cytometer. Current barcode strategy is based upon impregnating the plastic beads with fluorescent dyes with different wavelengths and different loading intensities. Theoretically, the use of 4 different dyes with 10 different intensities would result in 10,000 barcodes. However, the emission wavelengths of neighboring dyes cannot be too close together, or light from the emission shoulders will interfere with accurate intensity measurements. In practice, Luminox, Inc. offers a set of 100 beads made with ten loading intensities at two wavelengths. RMD proposes using a similar concept, but instead basing the molecular barcode on differences in fluorescent lifetime rather than intensity. Discrimination based on fluorescent lifetimes is not nearly as sensitive to interfering signal from neighboring dyes. The major component of radiative decay can easily be deconvoluted from minor, interfering components. This will allow dyes with emission maximums close togetherto be used resulting in a higher number of barcodes. In addition, because our approach is not susceptible to the errors inherent with absolute intensity measurements, less calibration will be required and possibly more than 10 variations at each wavelength will be distinguishable. If developed, this approach will have applications in disease research. For example, large population samples could studied for responses to therapeutic treatment as correlated with genetic variations.