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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）：BioHessen是哈佛医学院的Stanley塔博尔博士和Charles Richardson博士发明的新型基于引发酶的全基因组扩增（pWGA）技术商业化的独家许可方。该pWGA系统利用多种复制蛋白，包括引发酶/解旋酶、DNA聚合酶、单链DNA结合蛋白和几种其它辅助蛋白。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。I期研究的总体目标是确定将pWGA技术商业化以销售给研究人员的可行性。我们提出的一个关键方面是评估商业生产pWGA中使用的所有蛋白质组分的可行性。我们还将使用单核苷酸多态性（SNP）标记和微卫星标记的组合评估pWGA的性能，包括反应时间，产品收率以及扩增偏倚方面的产品质量。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的主要竞争者是多重置换扩增（MDA），其由GE Healthcare以GenomiPhi（tm）和Qiagen以RESID-g（r）销售。基于高信息微卫星标记的研究表明，MDA扩增与少量的输入DNA可能会导致基因组覆盖的损失。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在不损失基因组覆盖率的情况下扩增具有非常有限数量的输入DNA的样品的方法。实际上，pWGA使用几乎完整的复制体，因此当存在有限数量的初始拷贝时，可以在整个基因组上产生更均匀的扩增。此外，pWGA使用的酶也是复制型聚合酶，因此我们预期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遗传研究的有用工具。事实上，这些研究中使用的DNA样本往往数量有限。扩增整个基因组使研究人员能够对样本进行更多的测试。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）多重置换扩增（MDA）。Rubicon Genomics通过Sigma-Aldrich将GenomeTM试剂盒商业化用于研究。GE Healthcare以GenomiPhi（tm）的名称销售MDA技术，而Qiagen以RESID-g（r）的名称销售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扩增偏倚（例如，GC富集区）。MDA是基于噬菌体Phi 29 DNA聚合酶的链置换活性，并使用不同长度的随机寡核苷酸（通常为6-mer至8-mer）来引发DNA合成。MDA提供等温DNA扩增，其偏差小于基于PCR的方法。此外，Phi 29 DNA聚合酶提供了比Taq DNA聚合酶更大的复制保真度的希望，因为它是复制聚合酶。尽管如此，基于高度信息化的微卫星标记的研究表明，用少量输入DNA进行MDA扩增可能导致基因组覆盖率的损失。pWGA使用的酶也是复制聚合酶，因此我们预期pWGA将具有与MDA相当的复制保真度。此外，pWGA反应似乎比MDA快得多。在这个阶段，我们认为pWGA在扩增偏倚方面至少与MDA相当。我们在I期研究中获得的结果将有助于确定当使用少量模板DNA时pWGA是否具有较小的偏倚。