CIRCULATING DNA AMPLIFICATION & COLON CA DETECTION

描述(申请人提供)：这个为期两年的R21项目将证明，新开发的基因组扩增和分析技术将大大提高检测癌症患者血浆中肿瘤来源的循环核酸的灵敏度。特别是，我们开发了一种新技术，限制和环化辅助滚环扩增，RCA-RCA，它允许从经历了片段的DNA进行全面的全基因组扩增(即该方法对样品降解是容忍的)。因此，它可以扩增在人类血浆中循环的片段核酸，包括被用作早期肿瘤检测和癌症疾病监测的生物标记物的肿瘤衍生DNA。在一种改进的方案中，RCA-RCA还可以实现完整的甲基组扩增，即它保留了表观遗传变化，以便可以在扩增的材料中高通量地研究甲基化。我们的目标是证明，通过为高通量分子分析提供无限的材料，以及通过血浆循环DNA的微阵列分析允许高灵敏度和识别新的生物标记物，全基因组/甲基组扩增能够极大地改进对血浆中肿瘤起源的遗传或表观遗传学改变的检测。这项拟议的研究将聚焦于结肠癌。这项建议的前提是，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，往往无法检测到(S)。通过实现目标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方面实现极高的灵敏度和可靠性。此外，目前的全基因组扩增能够建立血浆DNA档案，使未来能够对相同的样本进行回溯性研究，以评估新的生物标志物并在研究人员之间共享材料。

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.