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，基于原始基因组基因组扩增（PWGA）技术的独家许可人。哈佛医学院的斯坦利·塔博尔（Stanley Tabor）和查尔斯·理查森（Charles Richardson）。该PWGA系统利用多种复制蛋白，包括原始酶/解旋酶，DNA聚合酶，单链DNA结合蛋白和其他几种辅助蛋白来实现快速敏感的整个基因组扩增。因此，将这项新型技术从大学实验室带到市场是一项艰巨的任务。在第一阶段，我们评估了该技术的性能，并在大生产规模上成功纯化了所有必要的蛋白质。我们成功启动了第一代RAPISOME“ PWGA套件，据我们所知，这是第一个从蜂窝再生的商业可用的DNA扩增系统重建。我们还发现了PWGA平台的其他优势功能。首先，我们已经显示PWGA平台可以实时检测DNA（100 fg）的实时可观，并证明了DNA（100 fg）。其他我们是否已进行了the，我们已经进行了。 （来自GE Healthcare的Genomiphi）PWGA平台在低拷贝数（100份）输入的圆形DNA上特别有效，这是II阶段研究的总体目标。启动）； 2）基于第1阶段的上述研究，在生物医学研究和临床诊断中探索了新的应用。根据I期数据，PWGA的应用可能会扩展到两个新领域。一种是作为通用DNA检测系统，将开发用于检测生物样品或药物产品中的痕量DNA污染。第二个新应用与圆形DNA扩增有关，基于I期观察结果，即PWGA可以以极高的效率扩增圆形DNA（从100份到超过1010份）。我们建议开发PWGA作为针对循环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扩增包含HPV的样品，然后使用Luminex系统确定HPV基因型。公共卫生相关性：整个基因组扩增（WGA）技术是用于癌症和遗传研究的有用工具。扩大整个基因组使研究人员能够对给定样本进行更多的测试，而不是其他可能的测试。其他两种类型的WGA已被商业化用于研究应用：1）从聚合酶链反应中得出的方法和2）多个位移放大（MDA）。 Rubicon Genomics已开发了一种基于PCR的WGA平台GenomePlex“用于研究用途的套件，可通过Sigma-Aldrich获得。GEHealthcare Market以Genomiphi的名称“ MDA技术”，而Qiagen以repledi-g（r）的名称出售MDA套件。这两种技术都有一些局限性。基因组”系统会产生短PCR扩增子，该系统限制了其在下游应用中的使用，例如RELP，DNA测序和克隆。此外，它需要进行多步反应设置。MDA通常还需要在等体性DNA放大之前进行热量启动启动启动启动的复杂性，并介绍该繁殖的复杂性，并介绍该繁殖的复杂性，并将其置于反应，并将其置于反应，并将其置于反应，该个段的反应，该突变是该集合的繁殖，该集合该集合，该突变，该群体，该繁殖的繁殖，该集合，该繁殖型，该繁殖的繁殖，该集合，该繁殖的繁殖，该集合能够构成。在没有输入模板的情况下，MDA中的随机底漆使其易于特异性的DNA扩增。并且可以将最小量（100 fg）的输入DNA的特定于输入DNA与背景区分开，这有可能被开发到一个给定样品中不需要的DNA污染的快速通用DNA检测工具中。与MDA相比，PWGA在放大圆形DNA方面效率更高100时，尤其是当输入量有限（100份）时。我们认为，PWGA的这一特征可用于开发高度敏感的多重分析，用于检测和基因分型，以检测和基因分型，例如人乳头瘤病毒（HPV）和单纯疱疹病毒（HSV）。