基于發(fā)卡型DNA自組裝及納米金比色一步法檢測核酸的新技術(shù)研究
發(fā)布時間：2018-12-07 08:17
【摘要】：核酸作為生命體重要的遺傳物質(zhì),它的檢測尤其重要,實現(xiàn)對核酸快速、簡單、可視化的檢測在生化分析領(lǐng)域具有重要的意義。無論是變溫的核酸擴增反應,還是等溫的核酸擴增反應,都是酶促反應,這類型的反應對實驗條件的高要求限制了核酸檢測的適用范圍。所以無酶DNA自組裝反應的提出,不僅在核酸檢測領(lǐng)域,同樣在DNA納米結(jié)構(gòu)和體內(nèi)檢測領(lǐng)域具有一定的應用價值。首先,本論文的第一章在無酶的指數(shù)發(fā)卡自組裝反應的基礎(chǔ)上,結(jié)合芘分子,展開指數(shù)發(fā)卡自組裝反應的熒光檢測,并對方法進行了驗證,但方案不能滿足核酸檢測的要求。因此,調(diào)整實驗方案,選用指數(shù)發(fā)卡自組裝反應結(jié)合熒光共振能量轉(zhuǎn)移對Cy3/Cy5,進行單鏈核酸的檢測。為獲得最佳的能量傳遞效率,對實驗條件進行了優(yōu)化。對方法的靈敏度進行的驗證結(jié)果表明,方法的檢測限為10 pM;利用添加10%胎牛血清的細胞培養(yǎng)基對方法的抗干擾能力進行檢測,結(jié)果表明方法具有很好的抗干擾能力;另外,通過對不同錯配靶標的檢測,證明了該方法具有良好的特異性。其次,本課題組的研究發(fā)現(xiàn),PEG 200能顯著提高鏈置換的速率,而DNA自組裝反應是DNA鏈之間自發(fā)的置換與雜交過程。因此我們設(shè)想PEG 200也可能促進DNA自組裝反應。在論文的第三章中,選取六種常用于核酸擴增的小分子物質(zhì),將它們用于雜交鏈式反應反應并比較了小分子物質(zhì)對雜交鏈式反應的影響,結(jié)果表明PEG 200為最佳的促進雜交鏈式反應的小分子物質(zhì),然后將PEG200用于指數(shù)發(fā)卡自組裝反應,結(jié)果顯示PEG 200同樣能促進指數(shù)發(fā)卡自組裝反應。而且,在20%的PEG 200的促進下,雜交鏈式反應的速率被提高100倍左右,指數(shù)發(fā)卡自組裝反應的速率被提高10倍左右。最后,論文的第四章以納米金比色為基礎(chǔ),從修飾后的納米金的抗鹽能力以及在加入靶標鏈后的變色時間及顏色變化情況比較了全修飾和不對稱修飾兩種納米金修飾方式,根據(jù)實驗結(jié)果確定全修飾的納米金適合用于核酸擴增反應。然后將全修飾的納米金用于聚合酶鏈式反應、環(huán)介導核酸等溫擴增反應中。結(jié)果顯示全修飾的納米金對核酸擴增反應有抑制作用,通過添加BSA可減少納米金對反應的抑制作用。但是實驗的比色結(jié)果表明,該方法還不能完成明顯的納米金比色。我們認為,可以通過優(yōu)化實驗條件,找到集納米金比色技術(shù)與核酸擴增反應于一體的一步核酸比色檢測法。
[Abstract]:Nucleic acid is an important genetic material in organism, and its detection is especially important. It is of great significance to detect nucleic acid quickly, easily and visually in the field of biochemical analysis. Both the reaction of nucleic acid amplification at varying temperature and the reaction of isothermal nucleic acid amplification are enzymatic reactions. The high requirements of these reactions limit the scope of application of nucleic acid detection. Therefore, the self-assembly reaction of non-enzymatic DNA not only in the field of nucleic acid detection, but also in the field of DNA nanostructure and in vivo detection has certain application value. In the first chapter of this paper, based on the enzymatic exponential card self-assembly reaction and pyrene molecule, the fluorescence detection of exponential card self-assembly reaction is carried out, and the method is verified, but the scheme can not meet the requirements of nucleic acid detection. Therefore, the single strand nucleic acid detection of Cy3/Cy5, was carried out by adjusting the experimental scheme and using exponential card self-assembly reaction and fluorescence resonance energy transfer. In order to obtain the best energy transfer efficiency, the experimental conditions were optimized. The test results show that the detection limit of the method is 10 pM; and the anti-interference ability of the method is detected by adding 10% fetal bovine serum to the cell culture medium. The results show that the method has good anti-interference ability. In addition, the detection of different mismatch targets proves that the method has good specificity. Secondly, our study shows that PEG 200 can significantly increase the rate of chain substitution, while the DNA self-assembly reaction is a spontaneous substitution and hybridization process between DNA strands. Therefore, we assume that PEG 200 may also promote DNA self-assembly reaction. In the third chapter of the thesis, six kinds of small molecules which are often used in nucleic acid amplification are selected and used in hybridization chain reaction and the effects of small molecules on hybridization chain reaction are compared. The results show that PEG 200 is the best small molecule to promote hybrid chain reaction, and then PEG200 is used in exponential card self-assembly reaction. The results show that PEG 200 can also promote exponential card self-assembly reaction. Moreover, with the promotion of 20% PEG 200, the rate of hybrid chain reaction was increased by about 100 times, and the rate of exponential hairpin self-assembly reaction was increased by about 10 times. Finally, the fourth chapter of the thesis based on nano-gold colorimetry, from the modified nano-gold salt resistance, after adding the target chain color change time and color changes, compared the full modification and asymmetric modification of two kinds of nano-gold modification methods. According to the experimental results, the fully modified gold nanoparticles are suitable for nucleic acid amplification. Then the fully modified gold nanoparticles were used in polymerase chain reaction and cyclized nucleic acid isothermal amplification reaction. The results showed that the fully modified gold nanoparticles could inhibit the nucleic acid amplification reaction, and the addition of BSA could reduce the inhibition effect of gold nanoparticles on the reaction. However, the experimental colorimetric results show that the method can not complete the apparent gold nanocrystalline colorimetry. We think that the one step nucleic acid colorimetry method can be found by optimizing the experimental conditions and combining the gold colorimetric technique with nucleic acid amplification.
【學位授予單位】：青島科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78
本文編號：2366893
[Abstract]:Nucleic acid is an important genetic material in organism, and its detection is especially important. It is of great significance to detect nucleic acid quickly, easily and visually in the field of biochemical analysis. Both the reaction of nucleic acid amplification at varying temperature and the reaction of isothermal nucleic acid amplification are enzymatic reactions. The high requirements of these reactions limit the scope of application of nucleic acid detection. Therefore, the self-assembly reaction of non-enzymatic DNA not only in the field of nucleic acid detection, but also in the field of DNA nanostructure and in vivo detection has certain application value. In the first chapter of this paper, based on the enzymatic exponential card self-assembly reaction and pyrene molecule, the fluorescence detection of exponential card self-assembly reaction is carried out, and the method is verified, but the scheme can not meet the requirements of nucleic acid detection. Therefore, the single strand nucleic acid detection of Cy3/Cy5, was carried out by adjusting the experimental scheme and using exponential card self-assembly reaction and fluorescence resonance energy transfer. In order to obtain the best energy transfer efficiency, the experimental conditions were optimized. The test results show that the detection limit of the method is 10 pM; and the anti-interference ability of the method is detected by adding 10% fetal bovine serum to the cell culture medium. The results show that the method has good anti-interference ability. In addition, the detection of different mismatch targets proves that the method has good specificity. Secondly, our study shows that PEG 200 can significantly increase the rate of chain substitution, while the DNA self-assembly reaction is a spontaneous substitution and hybridization process between DNA strands. Therefore, we assume that PEG 200 may also promote DNA self-assembly reaction. In the third chapter of the thesis, six kinds of small molecules which are often used in nucleic acid amplification are selected and used in hybridization chain reaction and the effects of small molecules on hybridization chain reaction are compared. The results show that PEG 200 is the best small molecule to promote hybrid chain reaction, and then PEG200 is used in exponential card self-assembly reaction. The results show that PEG 200 can also promote exponential card self-assembly reaction. Moreover, with the promotion of 20% PEG 200, the rate of hybrid chain reaction was increased by about 100 times, and the rate of exponential hairpin self-assembly reaction was increased by about 10 times. Finally, the fourth chapter of the thesis based on nano-gold colorimetry, from the modified nano-gold salt resistance, after adding the target chain color change time and color changes, compared the full modification and asymmetric modification of two kinds of nano-gold modification methods. According to the experimental results, the fully modified gold nanoparticles are suitable for nucleic acid amplification. Then the fully modified gold nanoparticles were used in polymerase chain reaction and cyclized nucleic acid isothermal amplification reaction. The results showed that the fully modified gold nanoparticles could inhibit the nucleic acid amplification reaction, and the addition of BSA could reduce the inhibition effect of gold nanoparticles on the reaction. However, the experimental colorimetric results show that the method can not complete the apparent gold nanocrystalline colorimetry. We think that the one step nucleic acid colorimetry method can be found by optimizing the experimental conditions and combining the gold colorimetric technique with nucleic acid amplification.
【學位授予單位】：青島科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78
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