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

基本信息

批准号：
7328472
负责人：
Huimin Kong
金额：
$ 11.06万
依托单位：
BIOHELIX CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2008-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7328472
关键词：
Bacteriophages Biological Assay Biomedical Research Clinical Condition DNA DNA Primase DNA amplification DNA biosynthesis DNA chemical synthesis DNA-Directed DNA Polymerase Development Diagnostic Enzymes GC Rich Sequence Generations Genetic Research Genome Genomics Genotype Goals Healthcare Healthcare Market Laboratories Length Marketing Measures Methods Microsatellite Repeats Names Nature Numbers Oligonucleotides Performance Phase Phase I Clinical Trials Polymerase Polymerase Chain Reaction Production Proteins Rate Reaction Reaction Time Research Research Personnel SS DNA BP Sales Sampling Short Tandem Repeat Single Nucleotide Polymorphism Staging System Taq Polymerase Technology Testing Universities anticancer research base commercialization design genetic selection helicase improved medical schools milligram new technology novel research study tool

项目摘要

DESCRIPTION (provided by applicant): BioHelix is the exclusive licensee for the commercialization of 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. As such, bringing this novel technology from the university laboratory to the market place is a challenging task. The overall goal of the Phase I research is to determine the feasibility of commercializing pWGA technology for sale to researchers. One of the key aspects we propose to evaluate in the feasibility of commercially producing of all of the protein components used in pWGA. We will also evaluate the performance of pWGA, including reaction time, product yield, as well as product quality in terms of amplification bias using a combination of single nucleotide polymorphism (SNP) markers and microsatellite markers. Finally, we will evaluate the replication fidelity of the pWGA technology using genetic selection methods. The leading competitor for pWGA is multiple displacement amplification (MDA) marketed by GE Healthcare as GenomiPhi(tm) and Qiagen as REPLI-g(r). Studies based on highly informative microsatellite markers suggest MDA amplifications with small quantities of input DNA can result in a loss of genome coverage. If we can successfully commercialize pWGA, we may be able to offer researchers with a means of amplifying samples with very limited quantities of input DNA without loss of genome coverage. Indeed, pWGA uses a virtually complete replisome and thus may yield a more uniform amplification on entire genomes when limited numbers of initial copies are present. Moreover, the enzymes used by pWGA are also replicative polymerases, therefore we expect pWGA will have a fidelity of replication that is equivalent to MDA. Finally, the pWGA reaction appears much more rapid than MDA. Whole genome amplification technologies are a useful tool for cancer research and genetic research. Indeed, DNA samples used in these studies are often available in limited quantities. Amplifying the entire genome enables researchers to perform more tests on the samples than would otherwise be possible. Two other types of WGA are currently being commercialized for research applications: 1) methods derived from the polymerase chain reaction and 2) multiple displacement amplification (MDA). Rubicon Genomics commercializes GenomePlex(tm) Kits for Research Use through Sigma-Aldrich. GE Healthcare markets MDA technology under the name GenomiPhi(tm) while Qiagen sells MDA kits under the name REPLI-g(r). A major problem of the PCR-based methods is incomplete coverage of the genome due to amplification bias of PCR reactions over certain loci (e.g., GC rich regions). MDA is based on the strand displacement activity of phage Phi29 DNA polymerase, and uses random oligonucleotides of varying lengths (typically 6-mer to 8-mer) to prime DNA synthesis. MDA offers isothermal DNA amplification with less bias that PCR based methods. In addition, Phi29 DNA polymerase offers the hope of greater fidelity of replication than Taq DNA polymerase because it is a replicative polymerase. Despite this, studies based on highly informative microsatellite markers suggest MDA amplifications with small quantities of input DNA can result in a loss of genome coverage. The enzymes used by pWGA is also a replicative polymerase, therefore we expect pWGA will have a fidelity of replication that is equivalent to MDA. In addition, the pWGA reaction appears much more rapid than MDA. At this stage, we believe pWGA is at least equivalent to MDA in terms of amplification bias. The results we would obtain during this Phase I study will help determine if pWGA has less bias when small quantities of template DNA is used.

描述（由申请人提供）：Biohelix是由DRS发明的小说，基于原始基因组的新颖的全基因组扩增（PWGA）技术的独家许可证。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。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。第一阶段研究的总体目标是确定将PWGA技术出售给研究人员的可行性。我们建议评估商业生产PWGA中所有蛋白质成分的可行性的关键方面之一。我们还将评估PWGA的性能，包括反应时间，产品产量以及产品质量在放大偏置方面，使用单核苷酸多态性（SNP）标记和微卫星标记的组合。最后，我们将使用遗传选择方法评估PWGA技术的复制保真度。 PWGA的主要竞争对手是GE Healthcare作为Genomiphi（TM）和Qiagen作为Repli-G（R）销售的多个位移扩增（MDA）。基于高度信息的微卫星标记的研究表明，具有少量输入DNA的MDA扩增会导致基因组覆盖率丧失。如果我们能够成功地将PWGA商业化，那么我们也许可以为研究人员提供一种可以放大数量有限的输入DNA的样品而不会损失基因组覆盖范围的方法。实际上，PWGA使用了几乎完整的重置体，因此当存在有限数量的初始副本时，可能会对整个基因组产生更均匀的扩增。此外，PWGA使用的酶也是复制性聚合酶，因此我们希望PWGA具有相当于MDA的复制性。最后，PWGA反应似乎比MDA快得多。整个基因组扩增技术是用于癌症研究和遗传研究的有用工具。实际上，这些研究中使用的DNA样品通常以有限的量提供。扩大整个基因组使研究人员能够对样品进行更多的测试，而不是其他可能的测试。目前，其他两种类型的WGA用于研究应用程序：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将基因组（TM）试剂盒商业化，以通过Sigma-Aldrich进行研究。 GE Healthcare Markets MDA技术名称为Genomiphi（TM），而Qiagen以Repli-G（R）的名义出售MDA套件。基于PCR的方法的一个主要问题是由于PCR反应在某些基因座上的放大偏置（例如GC富含区域），基因组的覆盖不完全。 MDA基于噬菌体PHI29 DNA聚合酶的链位移活性，并使用不同长度的随机寡核苷酸（通常为6-mer至8-mer）来素质量DNA合成。 MDA提供的等温DNA扩增和基于PCR的方法较少的偏差。此外，PHI29 DNA聚合酶比TAQ DNA聚合酶提供了更大的复制忠诚度，因为它是复制性聚合酶。尽管如此，基于信息丰富的微卫星标记的研究表明，具有少量输入DNA的MDA扩增会导致基因组覆盖率损失。 PWGA使用的酶也是复制性聚合酶，因此我们预计PWGA将具有相当于MDA的复制性。此外，PWGA反应似乎比MDA快得多。在此阶段，我们认为PWGA至少在放大偏见方面等同于MDA。在此阶段I研究中，我们将获得的结果将有助于确定当使用少量模板DNA时，PWGA是否具有较小的偏差。