Development of a novel whole genome amplification method that mimics nature

描述（由申请人提供）：BioHelix是一种新颖的、基于引物的全基因组扩增（pWGA）技术的独家许可人。哈佛医学院的Stanley Tabor和Charles Richardson。该pWGA系统利用多种复制蛋白，包括引物酶/解旋酶、DNA聚合酶、单链DNA结合蛋白和其他几种辅助蛋白，实现快速、灵敏的全基因组扩增。因此，将这项新技术从大学实验室推向市场是一项具有挑战性的任务。在I期，我们对该技术的性能进行了评估，并成功地在大规模生产中纯化了所有必需的蛋白质。我们成功推出了第一代Rapisome”pWGA试剂盒，据我们所知，这是第一个商业上可用的DNA扩增系统，由细胞复制体重建。我们还发现了pWGA平台的其他优势。首先，我们已经证明了pWGA平台可以实时检测微量DNA （100 fg）。此外，我们已经证明，与另一种商用WGA产品（GE Healthcare的GenomiPhi）相比，pWGA平台在从低拷贝数（100拷贝）的输入中扩增环状DNA（放大108倍）方面特别有效。第二阶段研究的总体目标将是：1)通过优化关键组件和微调每个组件来继续提高pWGA系统的性能（如果成功，将推出具有更低放大偏置和更高保真度的第二代pWGA套件）；2)在上述I期研究的基础上，探索pWGA在生物医学研究和临床诊断中的新应用。基于I期数据，pWGA的应用可能会扩展到两个新的领域。一是通用DNA检测系统，将用于检测生物样品或药品中痕量DNA污染。第二个新应用与环状DNA扩增有关，基于第一阶段的观察，pWGA可以以极高的效率扩增环状DNA（从100拷贝到1010拷贝以上）。我们建议将pWGA开发为针对人类乳头瘤病毒（HPV）等环状DNA病毒的临床研究和诊断工具。我们将使用pWGA扩增含有HPV的样本，随后使用Luminex系统确定HPV基因型。公共卫生相关性：全基因组扩增（WGA）技术是癌症和基因研究的有用工具。扩增整个基因组使研究人员能够在给定样本上进行比其他方法更多的测试。另外两种类型的WGA已经商业化用于研究应用：1)聚合酶链反应衍生的方法和2)多重位移扩增（MDA）。Rubicon Genomics开发了基于pcr的WGA平台GenomePlex“研究用工具包”，可通过Sigma-Aldrich获得。GE医疗以“GenomiPhi”的名称销售MDA技术，而Qiagen以“REPLI-g(R)”的名称销售MDA试剂盒。这两种技术都有一些局限性。GenomePlex系统产生短PCR扩增子，这限制了其在下游应用的使用，如RELP、DNA测序和克隆。此外，它需要一个多步骤的反应设置。在等温DNA扩增之前，MDA通常还需要一个热变性步骤，以促进引物退火，这增加了反应设置的复杂性，并可能在模板中引入突变。此外，在MDA中使用随机引物使其即使在没有输入模板的情况下也容易进行非特异性DNA扩增。引物碱基全基因组扩增（pWGA）平台是细胞复制体的体外重建。它执行一个简单的，一步等温全基因组扩增不添加引物。与PCR和MDA相比，pWGA反应速度更快。扩增对输入DNA模板具有高度的敏感性和特异性。少量（100 fg）输入DNA的扩增可以从背景中区分出来，这有可能发展成为一种快速的通用DNA检测工具，用于检测和定量给定样品中不需要的DNA污染。与MDA相比，pWGA扩增环状DNA的效率是MDA的100倍，特别是当输入量有限（100拷贝）时。我们相信，pWGA的这一特性可用于开发一种高灵敏度的多重检测方法，用于广谱环状DNA病毒的检测和基因分型，如人乳头瘤病毒（HPV）和单纯疱疹病毒（HSV）。

DESCRIPTION (provided by applicant): BioHelix is the exclusive licensee for a novel, primase-based Whole Genome Amplification (pWGA) technology invented by Drs. Stanley Tabor and Charles Richardson at Harvard Medical School. This pWGA system utilizes multiple replication proteins including a primase/helicase, a DNA polymerase, a single-stranded DNA binding protein, and several other accessory proteins to achieve rapid and sensitive whole genome amplification. As such, bringing this novel technology from the university laboratory to the market place is a challenging task. During Phase I, we evaluated the performance of this technology and successfully purified all necessary proteins in large production scale. We successfully launched the first generation Rapisome" pWGA kit, which is, to our knowledge, the first commercially available DNA amplification system reconstructed from a cellular replisome. We have also discovered additional advantageous features of the pWGA platform. First, we have shown that the pWGA platform can perform real-time detection of trace amounts of DNA (100 fg). In addition, we have demonstrated that in comparison with another commercially available WGA product (GenomiPhi from GE Healthcare) the pWGA platform is particularly efficient in amplifying (by 108 fold) circular DNA from low copy numbers (100 copies) of input. The overall goal of the Phase II research will be: 1) To continue to improve the performance of the pWGA system by optimizing the key components and fine-tuning each component (if successful, a second-generation pWGA kit with lower amplification bias and higher fidelity will be launched); 2) To explore new applications for pWGA in biomedical research and clinical diagnostics based on the aforementioned studies in Phase I. Based on the phase I data, the applications of pWGA may be expanded into two new areas. One is as a universal DNA detection system, which will be developed for the detection of trace amounts of DNA contamination in biological samples or pharmaceutical products. The second new application is related to circular DNA amplification, based upon the phase I observation that pWGA can amplify circular DNA with extremely high efficiency (from 100 copies to over 1010). We propose to develop pWGA as a tool for clinical research and diagnostic applications targeting circular DNA viruses such as human Papillomavirus (HPV). We will use pWGA to amplify samples containing HPV and subsequently determine the HPV genotype using the Luminex system. PUBLIC HEALTH RELEVANCE: Whole Genome Amplification (WGA) technologies are useful tools for cancer and genetic research. Amplifying the entire genome enables researchers to perform more tests on a given sample than would otherwise be possible. Two other types of WGA have been commercialized for research applications: 1) methods derived from the polymerase chain reaction and 2) multiple displacement amplification (MDA). Rubicon Genomics has developed a PCR-based WGA platform GenomePlex" Kits for Research Use, available through Sigma-Aldrich. GE Healthcare markets the MDA technology under the name GenomiPhi" while Qiagen sells MDA kits under the name REPLI-g(R). Both of these technologies have some limitations. The GenomePlex" system generates short PCR amplicons, which limits its use in downstream applications such as RELP, DNA sequencing, and cloning. In addition, it requires a multiple-step reaction setup. MDA also often requires a heat-denaturation step before isothermal DNA amplification to facilitate primer annealing, which increases the complexity of the reaction setup and may introduce mutations into the template. In addition, the use of random primers in MDA makes it prone to non-specific DNA amplification even in the absence of an input template. The primase-base Whole Genome Amplification (pWGA) platform is an in vitro reconstruction of a cellular replisome. It performs a simple, one-step isothermal whole genome amplification without added primers. The pWGA reaction is more rapid than either PCR or MDA. The amplification is highly sensitive and specific for input DNA template. Amplification of a minimal amount (100 fg) of input DNA can be distinguished from background, which has a potential of being developed into a fast universal DNA detection tool for detection and quantification of unwanted DNA contamination in a given sample. In comparison with MDA, pWGA is 100-times more efficient in amplifying circular DNA, especially when the amount of input is limited (100 copies). We believe that this feature of pWGA can be used to develop a highly sensitive multiplex assay for the detection and genotyping of a broad spectrum of circular DNA viruses, such as human Papillomavirus (HPV) and Herpes Simplex Virus (HSV).