SBIR Phase I: STR Analysis Using End-Labeled Free-Solution Electrophoresis

这个小企业创新研究（SBIR）的第一阶段研究项目将开发一种新的dna偶联标记（拖标），用于自由溶液电泳的基因片段大小分析。该方法不使用筛分基质，因此与使用纠缠聚合物溶液作为筛分基质的分离方式完全不同，并且具有更快的分离速度和更短的分离距离的优点。改进后的拖拽标签必须易于与DNA片段结合，并且具有足够低的大小差异，以便通过电荷而不是大小来分离DNA测序片段。该研究项目将对拖标进行测试，将其与PCR引物结合，用于短串联重复序列（STR）片段大小分析，并对其进行染料标记，用于荧光检测。我们将研究该方法的实用性，并对电泳分离性能进行评估，以确定限制分离效率的参数。此外，在不同大小片段的拖拽标签上改变染料分子数量后，基于相对强度的区分能力将被评估。商业上，应用是改进碎片大小分析使用自由溶液电泳。这种方法显著提高了分离速度和效率（降低成本），并在每个拖标上使用多个染料偶联物提高了灵敏度。在过去十年中，以纠缠聚合物溶液为筛分基质的毛细管电泳DNA测序技术取得了巨大的进展。新的测序技术有望提供更大的测序能力。然而，这些新的测序技术并不适用于基因片段大小分析方法，例如使用STR分析的法医人类身份和使用RFLP分析的其他应用。这些应用需要改进的技术。这种用于法医应用的自由溶液电泳方法可以整合到现有的毛细管电泳仪器中，从而实现快速和低成本的实施。

This Small Business Innovation Research (SBIR) Phase I research project will develop a novel DNA-conjugated marker (drag-label) in genetic fragment size analysis using free solution electrophoresis. This method uses no sieving matrix, so the separation occurs in a fundamentally different manner than using an entangled polymer solution as a sieving matrix and offers the advantage of faster separation with shorter separation distances required. The improved drag-labels must be readily conjugated to DNA fragments and have sufficiently low size variance to allow separation of the DNA sequencing fragments by charge rather than size. The research project will test a drag-label after it is conjugated to PCR primer for short tandem repeat (STR) fragment size analysis and dye-labeled for fluorescence detection. The utility of this method using the proposed drag-label will be investigated and electrophoresis separation performance evaluated to determine the parameters limiting separation efficiency. In addition, the ability to discriminate based on relative intensities after varying the number of dye molecules on the drag-labels of different sized fragments will be evaluated. Commercially, the application is improved fragment size analysis using free-solution electrophoresis. This approach offers significant improvement in separation speed and efficiency (cost reduction), with increased sensitivity using multiple dye-conjugates on each drag-label. DNA sequencing using capillary electrophoresis with an entangled polymer solution as the sieving matrix has experienced tremendous advances in the last decade. New technologies for sequencing are now promising even greater sequencing capabilities. However, these new sequencing technologies are not useful for genetic fragment size analysis methods, such as forensics human identity using STR analysis and other applications using RFLP analysis. Improved technologies are needed for these applications. This method for free-solution electrophoresis for forensics applications can be incorporated into existing capillary electrophoresis instrumentation, allowing rapid and low cost implementation.