- 學位論文
利用切口酶擴增反應連接滾環式擴增法檢測微核糖核酸
Combination of Nicking Enzyme Amplification Reaction and Rolling Circle Amplification for MicroRNA Detection
摘要
微核糖核酸（microRNA, miRNA, miR）是由約19-24核苷酸組成的RNA，因無法進一步轉譯成蛋白質，又稱為非編碼RNA，研究指出miRNA能調控基因表達，也跟許多癌症、疾病相關，對不同物種的影響程度也很大。Let-7是一種在不同物種間有高保留度的miRNA，在人類、果蠅、斑馬魚、老鼠都有let-7的蹤跡。本篇選擇果蠅的let-7進行實驗設計。由於miRNA有長度短、低豐度、易降解、同家族間相似度高等特色，導致精確檢測有困難，目前多數使用北方印跡（Northern blot）檢測果蠅體內的let-7。本篇嘗試連接內切酶擴增反應和滾環式擴增法兩種核酸擴增方法來檢測let-7，藉由兩步放大進而降低偵測極限，也期望提供新方法檢測果蠅體內的let-7或是其它miRNA。結果顯示LDR、NEAR、RCA皆能單獨進行，NEAR、RCA也能順利串聯，不過在LDR串聯NEAR時，遇到非特異性產物干擾，因而NEAR無法有效進行，後續將重新設計實驗細節，以改善產生非特異性產物。
並列摘要
MicroRNA (miRNA), approximately contain 19-24 nucleotides in length. This type of RNA cannot translate to proteins, hence they are also known as non-coding RNA. Research has indicated that miRNAs can regulate the expression of multiple genes. Abnormal expression of miRNAs is closely related to the occurrence, development, and spread of cancer and other diseases. miRNA exhibit significant effects across different species.Let-7 is one of the miRNAs that show highly conserved among different species, such as humans, drosophila, zebrafish, and mice. In this study, let-7 of drosophila was chosen for experimental design. Due to the characteristics of miRNAs, including their short length, low abundance, easy degradation, and high similarity within the family, accurate detection poses challenges. Currently, Northern blot is commonly used to detect let-7 in drosophila. This study was attempted to develop a Drosophila let-7 detection method through isothermal DNA amplification approaches. Two isothermal amplification methods (nicking enzyme amplification reaction, NEAR, and rolling circle amplification, RCA) were combined to minimize the interference of non-specific products generated by NEAR, and to obtain comparable LOD within a reasonable time of assay. Results showed that ligase detection reaction (LDR), NEAR, and RCA were able to be performed individually. NEAR and RCA were also successfully and successively carried out. However, the non-specific fragments produced by NEAR and LDR hampered the process of let-7 determination. Future works will focus on the designing of primer/probes sequences to reduce the non-specific products.