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、基于引物酶的全基因组扩增（pWGA）技术商业化的独家许可人。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。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。第一阶段研究的总体目标是确定将pWGA技术商业化并出售给研究人员的可行性。其中一个关键方面，我们建议评估的可行性，在商业生产中使用的所有蛋白质成分的pWGA。我们还将利用单核苷酸多态性（SNP）标记和微卫星标记的组合来评估pWGA的性能，包括反应时间、产品产量以及产品质量的扩增偏倚。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的主要竞争对手是多位移扩增（MDA），由GE Healthcare以GenomiPhi（tm）和Qiagen以REPLI-g(r)销售。基于信息丰富的微卫星标记的研究表明，少量输入DNA的MDA扩增可能导致基因组覆盖范围的丧失。如果我们能够成功地将pWGA商业化，我们可能能够为研究人员提供一种方法，在不损失基因组覆盖的情况下，用非常有限的输入DNA扩增样本。事实上，pWGA使用一个几乎完整的复制体，因此当有限数量的初始拷贝存在时，可能在整个基因组上产生更均匀的扩增。此外，pWGA使用的酶也是复制聚合酶，因此我们预计pWGA将具有与MDA相当的复制保真度。最后，pWGA反应似乎比MDA快得多。全基因组扩增技术是癌症研究和基因研究的有用工具。事实上，这些研究中使用的DNA样本通常数量有限。扩增整个基因组使研究人员能够对样本进行比其他方法更多的测试。另外两种类型的WGA目前正在商业化用于研究应用：1)来自聚合酶链反应的方法和2)多重位移扩增（MDA）。Rubicon Genomics通过Sigma-Aldrich将GenomePlex（tm）研究用试剂盒商业化。GE医疗以GenomiPhi（tm）的名称销售MDA技术，而Qiagen以REPLI-g(r)的名称销售MDA试剂盒。基于PCR的方法的一个主要问题是由于PCR反应在某些位点（例如GC富集区域）上的扩增偏倚而导致基因组的不完全覆盖。MDA是基于噬菌体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- 8-mer）来启动DNA合成。MDA提供等温DNA扩增与较少的偏见，PCR为基础的方法。此外，由于Phi29是一种复制聚合酶，因此它比Taq DNA聚合酶具有更高的复制保真度。尽管如此，基于信息丰富的微卫星标记的研究表明，少量输入DNA的MDA扩增可能导致基因组覆盖范围的丧失。pWGA使用的酶也是一种复制聚合酶，因此我们预计pWGA将具有与MDA相当的复制保真度。此外，pWGA的反应速度明显快于MDA。在这个阶段，我们认为pWGA在放大偏置方面至少相当于MDA。我们将在第一阶段研究中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。

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.