| 摘要: | 近年來液態切片（liquid biopsy）與生物分子診斷醫學分析興起，而常被醫學研究或臨床實驗上用來偵測病原體、遺傳性疾病或是癌症等病症。過程中，從各式組織樣品中能定量、定性及高品質地萃取生物分子如蛋白質、核酸等生物分子是作為精準醫療中的轉譯醫學非常重要的關鍵，也才足以作為後續生物分子診斷分析方法或設備的一個標準。如北方墨點法（Northern blot）、聚合酶連鎖反應 (Polymerase chain reaction)、核糖核酸陣列晶片(Nucleic acid Microarray assay)和次世代定序 (Next Generation Sequencing)等生物分子診斷，才足以提供不同診斷平台、診斷方式甚至是不同檢測執行單位之間的數據共享、整合與分析確認，進而得到更精準的檢測結果。本實驗室正在執行中的科技部計畫就是以研究各種商業產品（purification kits）以建立標準化之生物樣品純化微小核糖核酸（miRNA）為執行目標。計劃進行順利，也達預期目標。根據以上計劃之執行，我們發現不同商業核酸純化產品最終皆以二氧化矽管柱或纖維薄膜方式的固體表面吸附核酸做為主要及最終之純化作為。計畫執行中，同時也發現核酸與二氧化矽固體表面作用機制之研究卻也付之闕如。這也造成液態切片中核酸純化的標準化及作為轉譯醫學上很大的不確定性與可能發展。所以本計畫是利用本研究室長年以來深入研究的恆溫滴定微卡計（isothermal titration calorimetry, ITC）設備來探討核酸與二氧化矽固體表面之作用機制。探討之變數包括了：鹽類及濃度（鹽橋、氫鍵、水合作用）、pH值（質子化、帶電性）、溫度及液態切片中可能之生物雜質等。研究成果可以明確說明核酸藉由二氧化矽之純化機制，以提供液態切片最佳化及標準化的可能。 ;Nucleic acid detection from liquid biopsy for diagnosis and prognosis has gained intensive attention in the precision medicine paradigm and translation research. To quantitatively collect and qualitatively purified nucleic acids from tissue sample become imperative and inevitable steps before as inlet of any downstream biomolecular detection instruments, such as PCR, qPCR, microarray, NGS and any biosensor instruments for quantitation. Currently, most of the instrumental companies develop their own purification scheme and kits and is the most profitable business model. Arguments on minimum information and quality of sample preparation required for PCR/qPCR nucleic acid molecular detection has moved one step further for data collection for diagnosis and clinical purpose. However, no standard operation procedure (SOP) has reached among users and instruments developers. This is mainly due to that the complexity of the tissue sample and the mechanism of the purification scheme used nowadays is still not well understood. Our previous MOST funded project was to compare current used purification schemes and kits and to develop, possibly, a SOP for the translational clinical data portability among different platforms. We have successfully revealed the key differences among the kits under investigated and we also realized the complicated binding mechanism of using silica based column for nucleic acids purification, which is the mostly adapted scheme for final nucleic purification of the processes.Therefore, in this project, we would like to further study the binding mechanism from thermodynamics perspective of nucleic acids, including DNA and RNA, with silica surface by isothermal titration calorimetry (ITC). The size of the nucleic acids (RNA, mRMA, miRNA, DNA, gDNA, etc...), the type of salts and concentration (salt bridge, hydrogen bonding, hydration, etc…), the pH value (protonation of silica), the binding temperature and the contaminations from the lysis and biofluids are all the parameters under this investigation. The ITC mechanism studies of nucleic acids and silica surface should provide a clear understanding of the nucleic acids qualitative and quantitative purification and can serve as a meaningful translational indicator for molecular discovery, profiling, quantitation, validation and functional analysis. |
