Rapid and Efficient PCR Cleanup Filters

描述(由申请人提供)：DNA分析在许多应用中都很重要，包括取证、农业、病原体检测、诊断基因测试和生物医学研究。在绝大多数的基因组DNA分析中，为了增加研究中的遗传片段的数量，需要使用聚合酶链式反应(PCR)扩增靶序列等化学扩增方法，因此常用的分析方法的灵敏度是足够的。然而，对于一些重要的基因组分析，如毛细管测序、基于MALDI-TOF的基因分型、微阵列检测、限制性内切酶分析和克隆，聚合酶链式反应只是一个早期步骤。对于这些，通常需要对聚合酶链式反应产物进行“清理”，特别是去除未扩增的引物、多余的核苷酸(DNTP)和除所需扩增片段以外的其他有机成分。市场上的清理产品和方案对DNA分析所涉及的时间、成本和劳动力做出了重大贡献。对于相对较短的扩增片段(300个碱基对)，它们也表现出较差的DNA回收率，因为它们是首选的聚合酶链式反应速度和各种应用，例如多重聚合酶链式反应和处理受损的DNA或RNA。 迪菲尼蒂基因公司已经获得了罗彻斯特大学PI实验室开发的技术许可，该技术有望实现一步5分钟的聚合酶链式反应产物的一步清理，高通量的扩增产物，并有效地去除引物和dNTP。对于上述短扩增的情况，该方法具有特别的前景。这一分离过程利用了最近的一项发现，即固定在高表面积底物上的金纳米颗粒可以选择性地吸附短的单链DNA。我们的目标是利用这一观察结果，生产一种适用于上述应用的可商业化的原型套件。我们将对扩增产物、引物和dNTP的产量进行量化。 对用于固定金纳米颗粒的过滤器和表面附着化学的形状因数的具体改进将评估其在增加扩增片段产量和降低底物和dNTP产量方面的有效性。我们计划增加过滤器中使用的硅珠的表面积和金纳米颗粒的负载量，以使过滤器更快、更持久。我们还建议研究不带电荷的巯基硅烷和带负电的混合单层涂层的二氧化硅微珠，以减少过滤器中扩增子的损失。还将实施通过机械移除珠子来改进所需扩增DNA的回收。最后，我们安排了潜在客户对过滤器原型进行Beta测试。 个体遗传学将越来越多地被用于处方治疗过程和开发个性化药物，因此筛选和测序DNA对医学研究和诊断非常重要。聚合酶链式反应(PCR)产物通常用于DNA分析，但在测序之前需要进行漫长而繁琐的清理。我们建议将过滤器商业化，以提高从聚合酶链式反应产物中回收短DNA片段的效率，从而减少与清理过程相关的时间、成本和劳动力。

DESCRIPTION (provided by applicant): Analysis of DNA is important in many applications including forensics, agriculture, pathogen detection, diagnostic genetic testing and biomedical research. For the vast majority of genomic DNA analysis, chemical amplification methods such as polymerase chain reaction (PCR) amplification of a target sequence are used to increase the amount of the genetic fragment under study so that the sensitivity of common analytical methods is adequate. PCR, however, is only an early step for some important genomic assays such as capillary sequencing, MALDI-TOF based genotyping, microarray spotting, restriction analysis and cloning. For these, "clean up" of the PCR product is generally required, specifically to remove unamplified primers, excess nucleotides (dNTP) and additional organic components other than the desired amplicons. Cleanup products and protocols on the market are a significant contribution to the time, cost and labor involved in DNA analysis. They also exhibit poor recovery of DNA for relatively short amplicons (< 300 base pairs) as are preferred for speed of PCR and for a variety of applications such as multiplex PCR and handling of damaged DNA or RNA. Diffinity Genomics has licensed technology developed in the lab of the PI at the University of Rochester that is promising for single-step, 5 minute clean up of PCR product with high throughput for amplicons and efficient removal of primers and dNTP. The method has particular promise for the case of short amplicons cited above. The separation process exploits the recent finding that gold nanoparticles immobilized on high surface area substrates can selectively adsorb short, singlestranded DNA. Our goal is to take this observation and to produce a commercializable prototype kit suitable for the applications enumerated above. We will quantify the throughput of amplicons, primers and dNTP. Specific improvements to the form factor of the filter and surface attachment chemistry used to immobilize the gold nanoparticles will be evaluated for their effectiveness in increasing amplicon throughput and decreasing primer and dNTP throughput. We plan to increase surface area of silica beads utilized in the filters and the loading of gold nanoparticles in order to make the filters faster and longer lasting. We also propose to investigate uncharged mercaptosilane and negatively charged mixed monolayer coatings of the silica beads to reduce loss of amplicons in the filter. Improved recovery of the desired amplified DNA by mechanical removal of the beads will also be implemented. Finally, we have arranged for beta testing of the filter prototypes by prospective customers. Individual genetics will increasingly be used both to prescribe therapeutic courses and to develop personalized drugs so that screening and sequencing DNA are very important to medical research and diagnosis. Polymerase chain reaction (PCR) products are routinely used in DNA analysis but require lengthy and cumbersome cleanup prior to sequencing. We propose to commercialize filters that improve recovery of short DNA segments from PCR products as as reduce the time, cost and labor associated with the cleanup procedure.