【摘要】：DNA檢測(cè)在臨床疾病的診斷與治療、基因突變以及病原體檢測(cè)等方面具有重要意義,因而建立一種簡(jiǎn)單、快速、靈敏的檢測(cè)方法日益引人關(guān)注。近年,隨著生物化學(xué)和分子生物學(xué)的結(jié)合,電化學(xué)生物傳感器因其簡(jiǎn)單、易操作、響應(yīng)快、成本低等優(yōu)勢(shì)在生物分子檢測(cè)的研究中備受關(guān)注。為了提高檢測(cè)方法的靈敏度,核酸等溫?cái)U(kuò)增技術(shù)被研究發(fā)現(xiàn)并不斷發(fā)展。本研究整合兩種核酸等溫?cái)U(kuò)增技術(shù),利用電化學(xué)生物傳感器的檢測(cè)平臺(tái),對(duì)DNA進(jìn)行超靈敏檢測(cè)。本研究將循環(huán)鏈置換聚合反應(yīng)和超分支滾環(huán)擴(kuò)增兩種核酸等溫?cái)U(kuò)增技術(shù)相結(jié)合,構(gòu)建了一個(gè)超靈敏的電化學(xué)生物傳感器用于靶DNA的檢測(cè)。當(dāng)靶DNA存在時(shí),分子信標(biāo)與靶DNA特異性識(shí)別。兩者結(jié)合后分子信標(biāo)發(fā)生空間上的構(gòu)象改變,由莖環(huán)結(jié)構(gòu)變?yōu)殒湢罱Y(jié)構(gòu)。生物素標(biāo)記的引物與分子信標(biāo)暴露出來(lái)3’末端雜交,在d NTP和KF聚合酶的作用下延伸,引發(fā)循環(huán)鏈置換聚合反應(yīng)。循環(huán)鏈置換聚合反應(yīng)形成雙鏈DNA。這些雙鏈DNA有生物素標(biāo)記,之后通過(guò)生物素-親和素的結(jié)合作用將超分支滾環(huán)擴(kuò)增的引物和模板錨定在傳感器表面。在d NTP和phi29DNA聚合酶的作用下,超分支滾環(huán)擴(kuò)增反應(yīng)即可進(jìn)行并在電極表面產(chǎn)生大量的長(zhǎng)度不一的雙鏈DNA。最后,檢測(cè)探針與超分支滾環(huán)擴(kuò)增的產(chǎn)物雜交結(jié)合,然后通過(guò)生物素與親和素標(biāo)記的堿性磷酸酶結(jié)合,催化底物α-NP產(chǎn)生電信號(hào)。構(gòu)建的電化學(xué)生物傳感器可以對(duì)濃度0.01 f M~10 p M的靶物質(zhì)進(jìn)行簡(jiǎn)單、快速、定量檢測(cè),最低檢測(cè)限達(dá)到8.9 a M。特異性和靈敏性更高。該方法也可以用于其它DNA的檢測(cè)。在疾病的早期診斷和預(yù)后以及生物醫(yī)學(xué)研究方面也有很好的應(yīng)用前景。
[Abstract]:DNA detection plays an important role in the diagnosis and treatment of clinical diseases, gene mutation and pathogen detection. Therefore, the establishment of a simple, rapid and sensitive detection method has attracted more and more attention. In recent years, with the combination of biochemistry and molecular biology, electrochemical biosensors have attracted much attention in the research of biomolecular detection for their advantages of simple, easy to operate, fast response and low cost. In order to improve the sensitivity of detection methods, isothermal amplification of nucleic acid has been developed. In this study, two techniques of nucleic acid isothermal amplification were used to detect DNA by electrochemical biosensor. In this study, a highly sensitive electrochemical biosensor was constructed for the detection of target DNA by combining cyclic chain replacement polymerization and hyperbranching ring-amplification with two isothermal amplification techniques of nucleic acid. When the target DNA exists, the molecular beacons are specifically identified with the target DNA. The conformation of the molecular beacon changed from the stem ring structure to the chain structure. Biotin labeled primers and molecular beacons were exposed to 3 'end hybridization and extended under the action of d NTP and KF polymerase to initiate cyclic chain replacement polymerization. Cyclic chain displacement Polymerization to form Double-stranded DNA. These double-stranded DNA were labeled with biotin, and the primers and templates of hyperbranching and rolling amplification were anchored on the surface of the sensor by the combination of biotin and avidin. Under the action of d NTP and phi29DNA polymerase, the hyperbranching rolling ring amplification reaction can be carried out and a large number of double-stranded DNA. of different lengths can be produced on the electrode surface. Finally, the probe was hybridized with the product of hyperbranching roller amplification, and then the electrical signal was generated by the binding of biotin and affinity labeled alkaline phosphatase to catalyze the generation of electrical signal by 偽-NP. The electrochemical biosensor can be used to detect the target material with a concentration of 0.01 F M ~ (10) p ~ (-1). The detection limit is 8.9 am. Specificity and sensitivity are higher. This method can also be used for other DNA detection. It also has a good prospect in early diagnosis and prognosis of disease and biomedical research.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R440;TP212.3

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相關(guān)期刊論文 前1條

1 Jianjian Gao;Qingyun Zhang;Jianjun Xu;Lijuan Guo;Xuefeng Li;;Clinical significance of serum miR-21 in breast cancer compared with CA153 and CEA[J];Chinese Journal of Cancer Research;2013年06期

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本文編號(hào)：2382350