Development of a novel whole genome amplification method that mimics nature

开发一种模仿自然的新型全基因组扩增方法

• 批准号: 7608568
• 负责人: Huimin Kong
• 金额: $ 40.29万
• 依托单位: BIOHELIX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-12 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7608568
• 关键词: Archives; Area; Biological; Biological Assay; Biomedical Research; Circular DNA; Clinical; Clinical Research; Cloning; Condition; DNA; DNA Primase; DNA Sequence; DNA Viruses; DNA amplification; DNA-Directed DNA Polymerase; Data; Detection; Development; Diagnostic; Generations; Genetic Research; Genome; Genomics; Genotype; Goals; Healthcare; Healthcare Market; Heating; Human; Human Papillomavirus; In Vitro; Investigation; Kerckring' s valve; Knowledge; Laboratories; Marketing; Methods; Mutation; Names; Nature; Numbers; One-Step dentin bonding system; Papillomavirus; Performance; Pharmacologic Substance; Phase; Polymerase Chain Reaction; Production; Proteins; Public Health; Reaction; Reaction Time; Research; Research Personnel; SS DNA BP; Sales; Sampling; Simplexvirus; System; Technology; Testing; Time; Universities; base; cancer genetics; clinical application; helicase; improved; instrument; medical schools; new technology; novel; reconstruction; restoration; tool

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).

描述（由申请人提供）：BioHelix 是由 Drs. 发明的新型基于引物酶的全基因组扩增 (pWGA) 技术的独家授权商。哈佛医学院的斯坦利·塔博尔和查尔斯·理查森。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 结合蛋白和几种其他辅助蛋白，以实现快速、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。在第一阶段，我们评估了该技术的性能，并成功地大规模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，这是第一个由细胞复制体重建的商用DNA扩增系统。我们还发现了pWGA平台的其他优势特征。首先，我们证明了pWGA 平台可以对痕量 DNA (100 fg) 进行实时检测。此外，我们已经证明，与另一种市售 WGA 产品（来自 GE Healthcare 的 GenomiPhi）相比，pWGA 平台在检测方面特别有效。从低拷贝数（100 个拷贝）的输入中扩增（108 倍）环状 DNA 第二阶段研究的总体目标是： 1) 通过优化关键组件和精细化，继续提高 pWGA 系统的性能。调整每个组件（如果成功，将推出具有更低扩增偏差和更高保真度的第二代pWGA试剂盒）基于上述二期研究探索pWGA在生物医学研究和临床诊断中的新应用一、基于一期数据，pWGA的应用可能会扩展到两个新领域。一是作为通用DNA检测系统，将开发用于检测生物样品或药品中的痕量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个拷贝到超过1010个拷贝）。我们建议开发pWGA作为针对环状DNA病毒（例如人乳头瘤病毒（HPV））的临床研究和诊断应用的工具。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对给定样本进行比其他方式更多的测试。另外两种类型的 WGA 已商业化用于研究应用：1) 源自聚合酶链式反应的方法和 2) 多重置换扩增 (MDA)。 Rubicon Genomics 开发了基于 PCR 的 WGA 平台 GenomePlex“研究用试剂盒”，可通过 Sigma-Aldrich 购买。GE Healthcare 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”系统生成短 PCR 扩增子，这限制了其在 RELP、DNA 测序和克隆等下游应用中的使用。此外，它需要多步骤反应设置。MDA 在等温 DNA 之前通常还需要一个热变性步骤扩增以促进引物退火，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 中使用随机引物使得即使在没有输入的情况下也容易发生非特异性 DNA 扩增。基于引物酶的全基因组扩增 (pWGA) 平台是细胞复制体的体外重建，无需添加引物即可执行简单的一步式等温全基因组扩增。 pWGA 反应比 PCR 或 MDA 更快。扩增对输入 DNA 模板具有高度敏感性和特异性，可以将最小量（100 fg）的输入 DNA 扩增与背景区分开，这有可能被开发为用于检测和检测的快速通用 DNA 检测工具。定量给定样品中不需要的 DNA 污染。与 MDA 相比，pWGA 扩增环状 DNA 的效率高出 100 倍，特别是当输入量有限（100 个拷贝）时。我们相信，pWGA 的这一特性可用于开发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，例如人乳头瘤病毒 (HPV) 和单纯疱疹病毒 (HSV)。