應用磷酸根甲基化去氧核醣核酸引子以提升檢測單一核酸變異和微核醣核酸專一性之研究;Studies of improving detection specificity of single nucleotide variation and microRNAs by phosphate-methylated DNA primers

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题名:	應用磷酸根甲基化去氧核醣核酸引子以提升檢測單一核酸變異和微核醣核酸專一性之研究;Studies of improving detection specificity of single nucleotide variation and microRNAs by phosphate-methylated DNA primers
作者:	洪靖雅;Hung, Ching-Ya
贡献者:	化學工程與材料工程學系
关键词:	磷酸根甲基化去氧核醣核酸;聚合?鏈鎖反應;引子設計;微核醣核酸;單一核酸變異;核酸類似物;neutralized DNA;phosphate-methylated DNA;single nucleotide variants;microRNA;Polymerase chain reaction;primer design
日期:	2021-09-08
上传时间:	2021-12-07 11:33:28 (UTC+8)
出版者:	國立中央大學
摘要:	隨著科技的進步，基因檢測已被廣泛用來協助醫生進行臨床疾病的篩檢及診斷，並對患者建立個人化治療。聚合?鏈反應(PCR)具有的檢測靈敏度和精確性，在臨床醫療上作為基因診斷的標準檢測工具。在PCR檢測中遇到單核?酸多態性(SNP)、單核?酸變異(SNV)或是具有序列高度相似性的微小RNA (microRNA)時，容易因為分辨效果不佳而造成錯誤的診斷，影響治療成效。因此，有許多研究將DNA和RNA核酸類似物改質到檢測目標序列的引子上，以提升PCR檢測的精準性。磷酸根甲基化DNA (nDNA)是由本實驗室所發展的一種核酸類似物，因為磷酸骨架被修飾上甲基基團，使nDNA單體成為電中性。將nDNA修飾在引子序列上可降低引子與欲檢測的核酸模板上雙股間的靜電排斥力，增加雙股的穩定性，但同時也因為甲基基團在雙股形成時也會產生立體障礙，降低雙股結構的穩定性。本研究藉由調控引子上nDNA的改質位置及數量，期望獲得檢測專一性最佳的nDNA修飾引子。本研究中使用部分修飾nDNA的引子，藉由調控nDNA於引子上的位置，以提升即時聚合?鏈鎖反應(qPCR)及反轉錄即時聚合?鏈鎖反應(RT-qPCR)對DNA及miRNA的檢測專一性。在檢測具有SNV的qPCR實驗中，使用KRAS基因作為本研究之目標序列，並透過調整不同nDNA的修飾位置，來增加nDNA修飾引子對SNV的辨識能力。而在RT-qPCR實驗中，使用僅有單鹼基差異的let-7a及let-7c作為目標miRNA，並透過將nDNA修飾在stem-loop反轉錄引子上不同的位置，分辨高度序列相似的miRNA。由實驗結果證實，在區分SNV的qPCR實驗中，藉由調控nDNA修飾位置以及引子與模板在qPCR操作時的黏合溫度（退火溫度），可以讓nDNA修飾引子的辨識能力明顯優於未修飾nDNA的引子。從擴增動力學方面來看，nDNA修飾引子與wild-type(WT)模板的擴增效率與未修飾nDNA的引子相似，但相較未修飾nDNA的引子能更有效抑制SNV序列的擴增。最後RT-qPCR的實驗結果顯示，nDNA修飾之反轉錄引子在辨識高度序列相似性miRNA的能力明顯優於未修飾nDNA的反轉錄引子，並可藉著調控nDNA的修飾位置影響反轉錄引子的結構及反轉錄?的活性，得到檢測專一性最佳之反轉錄引子。本研究已成功將nDNA的修飾序列應用於qPCR以及RT-qPCR中，並可藉由引子上nDNA的設計及操作條件提升PCR檢測的專一性。期望未來通過合適的序列設計及nDNA修飾位置，將nDNA修飾序列應用在各種核酸檢測平台，以提供醫療上更精準的診斷及治療。 ;With advanced technologies, genetic testing has been widely used to assist clinicans in diseases screening and diagnosis as well as establishing personalized treatment for patients. Polymerase chain reaction (PCR) with high sensitivity and accuracy is considered a standard method for detection and diagnosis of genetic abnormalities in clinical medicine. However, encountering single nucleotide polymorphism (SNP), single nucleotide variation (SNV) or microRNAs (miRNA) with high sequence similarity easily falsify PCR results, affecting diagnosis and treatment efficacy. Therefore, many studies have used nucleic acids analogues or derivatives to be target sequence primers to improve the accuracy of PCR detection. Phosphate-methylated DNA (designated as neutralized DNA or nDNA) is a nucleic acid analog created by modifying the phosphate backbone with methyl groups to electrically neutralize the DNA monomer. Modifying the nDNA on the primer therefore can reduce the electrostatic repulsion between the primer and the aimed template of the double strands, increasing the stability of the double strands. However, these methyl groups also cause electrosteric effects on the formation of double strands, destabilizing the duplex. In this study, by modifing the positions and quantity of nDNA on the primer, we are expect to obtain the nDNA modified primer with the wishes detection specificity. In this study, partially nDNA-modified primers were used. By adjusting the modified positions of nDNA on the primers, both specificity in DNA detection by real-time Polymerase chain reaction (qPCR) and miRNA detection by Reverse transcription real-time polymerase chain reaction (RT-qPCR) were improved. In the qPCR detection for SNV, the KRAS gene was need as the target sequence, and the ability of nDNA-modified primers to recognize SNV was enhanced by varied the nDNA-modified sites. Additionally, in determining miRNAs by RT-qPCR, let-7a and let-7c with only one single base difference served as target miRNAs, and reverse transcription primers with diversified nDNA-modified locations were used to discriminate these highly similar sequence miRNAs. The experimental results confirmed that in distinguishing SNV by qPCR, adjusting the nDNA-modified position and the binding temperature (annealing temperature) of the primer and template significantly increased its identification compared to that of unmodified primer. From the perspective of amplification kinetics, compare with unmodified primer, the modified nDNA primer achieved similar PCR amplification with the perfect match template but more effectively inhibit the amplification of SNV sequence. Finally, the experimental results of RT-qPCR showed that the ability of the nDNA modified reverse transcription primer to discriminate miRNAs with high sequence similarities was significantly better than that of the unmodified one. Additonally, manipulating the modified position of the nDNA can affect the primer structure and the activity of reverse transcription enzyme, therefore optimizing detection specificity of the reverse transcription primer. At present, the nDNA designed sequence has been successfully applied to qPCR and RT-qPCR primer modification. Moreover, appropriate primer design and operating conditions can improve the qPCR and RT-qPCR specificity. Hopefully in the future, through proper sequence design and nDNA modification positions, nDNA-modified sequences can be applied to various nucleic acid detection platforms to provide more accurate medical diagnosis and treatment.
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