Thermostable primer-free whole genome amplification of single cells with viral en

使用病毒核酸对单细胞进行热稳定性无引物全基因组扩增

• 批准号: 8591681
• 负责人: Baigen Mei
• 金额: $ 15万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8591681
• 关键词: Area; Cancer Biology; Cardiology; Cells; DNA; DNA Primase; DNA amplification; DNA biosynthesis; DNA-Directed DNA Polymerase; Development; Disadvantaged; Disease; Dropout; Escherichia coli; Evaluation; Genetic; Genome; Genomics; Genotype; Goals; Government; High temperature of physical object; Human; Human Microbiome; Human body; Libraries; Link; Maps; Measures; Methods; Microbial Biofilms; Microbiology; Morphologic artifacts; Neurology; Nucleotides; Oligonucleotides; Persons; Phase; Phenotype; Polymerase; Proteins; Protocols documentation; Reaction; Reagent; Research; Rhodobacter sphaeroides; Sample Size; Sampling; Sequence Analysis; Side; System; Technology; Variant; Viral; Work; base; cancer cell; cancer stem cell; expression cloning; improved; microbial genome; next generation sequencing; novel; public health relevance; viral DNA

DESCRIPTION (provided by applicant): The genetic basis of disease remains a significant research problem, impacting the fields of cancer biology, neurology, cardiology, development, and even microbiology, as recently realized by the Human Microbiome Project. Determination of the genomic sequence underlying observed phenotypes is now widespread. Genomic sequencing is becoming faster and cheaper, allowing access to more genomic information and greater understanding of the relationships between genotype and phenotype. Increasingly, these technologies are being applied to smaller and smaller sample sizes, down to single cells. A single cell contains all the necessary genetic information, and theoretically can be amplified repeatedly. However, generating enough amplified DNA from such small samples for genomic sequencing is constrained by several features of current technologies. Current amplification protocols use phi29 polymerase and DNA primed with random hexamers. Although this system has allowed for the study of disease using single cells, it has numerous disadvantages. Primary among these are primer amplification artifacts and high bias which is especially exacerbated when the amount of target DNA is low. Biased amplification from single genomes results in loss of sequence information such as allelic dropout and complicates copy number variant analysis. This bias is stochastic because it results from the initial annealing of random hexamers to begin amplification from random regions. Such stochastic bias makes comparison between single cells nearly impossible. The goal of this Phase I proposal is to enable primer-free DNA synthesis, to vastly improve the efficiency and coverage of amplified DNA from samples as small as single cells. Removing primers will eliminate unproductive side-reactions and decrease bias in the final product. This system is expected to operate isothermally at higher temperatures, without the need for an initial denaturation step. The commercial result of our proposed study will be a reagent kit for high fidelity, low bias (high coverage) amplification of DNA from single cells without primer background issues. This work will advance the study of single cell genomics by providing higher sequence coverage, enabling research into cancer, stem cells, biofilms, and the human microbiome.

描述(申请人提供)：疾病的遗传基础仍然是一个重要的研究问题，影响到癌症生物学、神经学、心脏病学、发育甚至微生物学领域，最近由人类微生物组项目实现。对观察到的表型背后的基因组序列的确定现在很普遍。基因组测序变得更快、更便宜，这使得人们能够获得更多的基因组信息，并更好地了解基因和表型之间的关系。这些技术越来越多地被应用于越来越小的样本量，小到单个细胞。单个细胞包含所有必要的遗传信息，理论上可以重复扩增。然而，从如此小的样本中产生足够的扩增DNA用于基因组测序受到当前技术的几个特征的限制。目前的扩增方案使用phi29聚合酶和随机六聚体启动的DNA。虽然这一系统允许使用单细胞研究疾病，但它有许多缺点。其中最主要的是引物扩增伪影和高偏倚，当目标DNA含量较低时，这一点尤其严重。单个基因组的偏向扩增会导致等位基因丢失等序列信息的丢失，并使拷贝数变异分析复杂化。这种偏向是随机的，因为它是从随机区域开始扩增的随机六聚体的初始退火法的结果。这种随机偏差使得单个细胞之间的比较几乎是不可能的。这一第一阶段提案的目标是实现无引子的DNA合成，极大地提高从小到单细胞的样本中扩增DNA的效率和覆盖率。去掉底物将消除无效的副反应，并减少最终产品中的偏差。该系统预计将在更高的温度下等温运行，而不需要初始变性步骤。我们提议的研究的商业化结果将是一种试剂盒，用于高保真、低偏差(高覆盖率)的单细胞DNA扩增，而不存在引物背景问题。这项工作将通过提供更高的序列覆盖率来推进单细胞基因组学的研究，使对癌症、干细胞、生物膜和人类微生物组的研究成为可能。