- 學位論文
焦磷酸定序法之改善及應用
Improvement and application of the pyrosequencing.
摘要
焦磷酸定序法為新世代定序之一，其原理為DNA聚合酶(DNA polymerase)聚合相對應核甘酸至模板後，會釋放焦磷酸(pyrophosphate)，利用酵素將焦磷酸(pyrophosphate)轉成冷光訊號。 本論文研究分兩部份，一部份為改善焦磷酸定序法。焦磷酸定序帄均長度為300個鹼基對，其中一個原因可能是引子流失造成。為了改善引子流失率，我們利用兩種引子來實驗。一組為一般使用的引子稱為習知引子；另一組是有髮夾狀結構的引子，利用連結酵素將模板與此引子連接，則該待測之單股DNA形成類似雙股DNA互補的結構，稱為可自我黏合引子。利用這兩種引子來進行清洗步驟，並量測引子流失率。NAD-dependent連接酵素在進行連接反應時會釋放AMP，經由定量AMP來量測習知引子、自我黏合引子流失的情形。習知引子帄均流失率為29.4%±6.5%，自我黏合引子帄均流失率為-0.03%±2.3%。當鹽濃度越低，則習知引子流失率越高，而我們製備的自我黏合引子，自我黏合引子的流失率不會受到鹽濃度所影響。 另一部分，本論文將焦磷酸定序法應用於測量microRNA。以microRNA當作DNA複製時的RNA引子，設計DNA模版，其與microRNA互補，兩者雜交後，DNA聚合酶將適當的核苷酸聚合至microRNA上，同時產生焦磷酸。使用人工合成之mir-21 RNA，測量範圍為5fmol至200fmol並做出標準曲線，其相關係數為0.998。測量SiHa 細胞之mir-21含量，在RNA總量為30μg時，三次測量帄均為18.1 ±4.2fmol。
並列摘要
Pyrosequencing is one of the next generations for DNA sequencing. The principle of this method is to detect pyrophosphate released from the incorporation of nucleotides with the DNA primer during the DNA synthesis catalyzed by DNA polymerase. There are two parts in this study. The first part is to improve pyrosequencing. The average reading-length for pyrosequencing is about 300 nucleotides. We assumed the primer loss is one of the reasons for the reading-length limit because there are washing steps during pyrosequencing and the hydrogen bonds, the binding force between the two strands of DNA, are week. To prove this assumption and to improve the primer-loss rate, we designed two primers. One is a stem-loop primer and another is a general primer. The stem-loop primer was ligated with its template. After washing, the average loss rates were 29.4%±6.5% and -0.03%±2.3% for the general primer and the stem-loop primer, respectively. We also studied their loss rates in different concentrations of salt. The lower salt concentration, the higher general-primer loss-rate. On the other hand, the loss rate of the stem-loop primer was not affected by salt concentration. The second part is to apply pyroseqencing in microRNA detection.The principle is to use microRNA as RNA primer during the DNA synthesis. The hsa-miR-21, a microRNA in human cell, was used as the model microRNA and a DNA template was designed to hybridize with it. This method has a linear detection range of 5 to 200 femtomoles under conditions used. The correlation coefficient was 0.998. The miR-21 level in 30μg total RNA from SiHa cells extraction was quantified and the average of three measurements was 18.1 ± 4.2fmol.