CIRCULATING DNA AMPLIFICATION & COLON CA DETECTION

描述（由申请人提供）：这项为期2年的R21项目将证明，新开发的基因组扩增和分析技术可大大提高检测癌症患者血浆中肿瘤来源循环核酸的灵敏度。特别是，我们开发了一种新技术，限制性和环化辅助滚环扩增，RCA-RCA，允许从经历片段化的DNA进行全面的全基因组扩增（即该方法对样品降解具有耐受性）。因此，它可以扩增在人血浆中循环的片段化核酸，包括用作癌症早期肿瘤检测和疾病监测的生物标志物的肿瘤来源的DNA。在修改的方案中，RCA-RCA还可以实现全甲基化组扩增，即，它保留了表观遗传变化，使得可以在扩增的材料中以高通量为基础研究甲基化。我们的目的是证明全基因组/甲基化组扩增通过为高通量分子分析提供无限的材料和通过允许高灵敏度和经由血浆循环DNA的微阵列分析鉴定新的生物标志物，使得能够高度改进血浆中肿瘤来源的遗传或表观遗传改变的检测。这项拟议的研究将集中在结肠癌。该建议的前提是，在早期结肠癌中，肿瘤DNA在血浆中循环，但由于检测方法和可用材料的限制，通常未被检测到。通过实现“靶放大”和高通量基因组分析的应用，我们的目标是在检测血浆中肿瘤来源的循环核酸中实现高度提高的灵敏度和可靠性。此外，本发明的全基因组扩增使得能够建立血浆-DNA档案，这使得能够对相同样品进行未来的回顾性研究，以用于评价新的生物标志物和在研究者之间共享材料。

DESCRIPTION (provided by applicant): This 2-year R21 project will demonstrate that newly developed genome amplification and analysis technologies lead to highly improved sensitivity in detecting circulating nucleic acids of tumor origin in the plasma of patients with cancer. In particular, we have developed a new technology, Restriction and Circularization-Aided Rolling Circle Amplification, RCA-RCA that allows comprehensive whole genome amplification from DNA that has undergone fragmentation (i.e. the method is tolerant to sample degradation). As such, it can amplify fragmented nucleic acids circulating in human plasma, including tumor-derived DNA that is used as a biomarker for early tumor detection and disease monitoring in cancer. In a modified protocol, RCA-RCA can also achieve whole methylome amplification, i.e., it preserves epigenetic changes so that methylation can be studied on a high throughput basis in the amplified material. We aim to demonstrate that whole genome/methylome amplification enables highly improved detection of tumor-derived genetic or epigenetic alterations in blood plasma by providing unlimited material for high- throughput molecular analysis and by allowing high sensitivity and identification of new biomarkers via microarray analysis of plasma-circulating DNA. The proposed study will focus on colon cancer. The premise of this proposal is that, in early colon cancer, tumor DNA is circulating in the plasma but often goes undetected due to limitations in the detection method(s) and the material available. By enabling 'target magnification' and application of high-throughput genome analysis we aim to achieve highly improved sensitivity and reliability in detecting circulating nucleic acids of tumor origin in plasma. Furthermore, the present whole genome amplification enables plasma-DNA archives to be established that enable future retrospective studies to be conducted on the same samples for evaluation of new biomarkers and sharing of material among investigators.