基于G四聯(lián)體DNA模擬酶和接合探針的核酸檢測新方法研究
發(fā)布時(shí)間：2019-04-24 19:01
【摘要】：G四聯(lián)體(G4)是一種特殊的核酸二級結(jié)構(gòu),其由富含鳥嘌呤堿基(G)的寡核苷酸序列通過Hoogsteen鍵連接而成。在含有一些單價(jià)金屬離子的溶液中,G4可與氯化血紅素單體(Hemin)結(jié)合形成具有過氧化物酶活性的功能性寡核苷酸結(jié)構(gòu),又被稱為 DNA 模擬酶(peroxidase-mimicking DNAzyme)。利用 DNA 模擬酶作為信號檢測元件對于核酸檢測具有極強(qiáng)的便攜性,然而現(xiàn)今基于DNA模擬酶的核酸均相檢測方式具有較大的檢測背景,從而阻礙了 DNA模擬酶在核酸均相檢測中的廣泛應(yīng)用。接合探針(junction probe,JP)源于雙探針概念(binary probe,BP),在一個(gè)系統(tǒng)中,當(dāng)且僅當(dāng)兩條探針同時(shí)與靶核酸序列雜交時(shí)可產(chǎn)生雜交信號,使其具有極強(qiáng)的雜交特異性,并且有潛力被運(yùn)用于核酸的均相檢測。傳統(tǒng)的接合探針由于有較大的非特異性雜交概率,而使其需要經(jīng)過復(fù)雜的序列設(shè)計(jì)及優(yōu)化,這增加了檢測的成本。本研究在前人研究的基礎(chǔ)上,通過構(gòu)建基于鏈置換技術(shù)(strand displacement)的接合探針,并整合G四聯(lián)體用于核酸的便攜檢測,并初步評價(jià)其檢測效能。論文主要分為以下兩個(gè)章節(jié):在第一章的研究中,我們將分裂的G-四聯(lián)體形成序列整合于兩個(gè)探針中。只有在含有特異性可識別剪接位點(diǎn)的靶基因轉(zhuǎn)錄本存在時(shí),探針可相互組裝形成三向接合構(gòu)象,從而提供功能性的G-四聯(lián)體構(gòu)象,其與氯化血紅素單體結(jié)合后可大大增強(qiáng)氯化血紅素的過氧化酶活性。該系統(tǒng)對通過比色測定對靶基因轉(zhuǎn)錄本的檢測限為0.063 μM,并且可特異地識別靶序列中的單堿基突變(約需3倍量的突變序列才能使檢測體系獲得與靶序列等同的信號)。該接合探針識別的靶序列可長達(dá)46 bp,并且無需復(fù)雜的序列設(shè)計(jì)步驟,顯示出其用于進(jìn)一步構(gòu)建簡便、高效的核酸檢測系統(tǒng)的潛力。第二章中,我們構(gòu)建了一種用于核酸靶序列識別和信號放大的方法。核酸擴(kuò)增基于鏈置換擴(kuò)增技術(shù)(strand displacement amplification,SDA),其中兩條 DNA 探針在抑制鏈(inhibitor)作用下不相互組裝觸發(fā)下游鏈置換擴(kuò)增。但在靶序列存在時(shí),探針可通過靶序列誘導(dǎo)的三向接合結(jié)構(gòu)形成(核心原理見第一章節(jié))釋放抑制鏈,隨后觸發(fā)SDA反應(yīng)擴(kuò)增富C(胞嘧啶)序列模板,這導(dǎo)致一系列富G(鳥嘌呤)序列生成,其與氯化血紅素單體結(jié)合表現(xiàn)出強(qiáng)大的過氧化物酶活性。用TMB作為底物并將產(chǎn)物在450nm的波長下測定時(shí)時(shí),其具有0.8 pM的檢測限。該檢測系統(tǒng)具有極低的檢測背景以及核酸檢測的高選擇性。
[Abstract]:G-tetraad (G-4) is a special secondary structure of nucleic acid, which is composed of oligodeoxynucleotide sequences rich in guanine base (G) through Hoogsteen bonds. In solutions containing some monovalent metal ions, G4 binds to the hemin monomer (Hemin) to form a functional oligodeoxynucleotide structure with peroxidase activity, also known as the DNA mimicase (peroxidase-mimicking DNAzyme). Using DNA mimetic enzyme as signal detection element is very portable for nucleic acid detection. However, nowadays, homogenous detection of nucleic acid based on DNA mimetic enzyme has a large detection background. This hinders the wide application of DNA mimetic enzyme in nucleic acid homogenous detection. Conjugation probe (junction probe,JP) is derived from the concept of double probe (binary probe,BP). In a system, if and only if two probes are hybridized with the target nucleic acid sequence at the same time, the hybridization signal can be generated, which makes it very specific for hybridization. And it has the potential to be used for homogenous detection of nucleic acids. Because of the large non-specific hybridization probability, the traditional conjugation probe needs complex sequence design and optimization, which increases the cost of detection. In this study, based on the previous studies, the conjugation probe based on the chain substitution technique (strand displacement) was constructed, and the G-tetraad was integrated into the portable detection of nucleic acid, and its detection efficiency was evaluated preliminarily. The thesis is divided into two chapters: in the first chapter, we integrate the split G-tetraad formation sequence into two probes. Only in the presence of transcripts containing specific splicing sites, probes can be assembled together to form a three-way conjugation conformation, thus providing a functional G-tetraad conformation. After binding with hemin monomer, the peroxidase activity of hemin can be greatly enhanced. The detection limit for target gene transcripts by colorimetric assay is 0.063 渭 M, and the system can specifically identify single base mutations in the target sequence (about 3 times the amount of mutation sequence is required for the detection system to obtain the same signal as the target sequence). The target sequence identified by the conjugation probe can be up to 46 bp, long and does not require complex sequence design steps, showing its potential for further construction of a simple and efficient nucleic acid detection system. In the second chapter, we construct a method for nucleic acid target sequence recognition and signal amplification. Nucleic acid amplification is based on chain substitution amplification (strand displacement amplification,SDA), in which two DNA probes do not assemble each other to trigger downstream strand substitution amplification under the action of inhibitory chain (inhibitor). However, in the presence of the target sequence, the probe can release the inhibitory chain through the three-way conjugation structure induced by the target sequence (see chapter I for the core principle), and then trigger the SDA reaction to amplify the C (cytosine)-rich sequence template. This leads to the formation of a series of G-rich (guanine) sequences, which combine with hemin monomers and exhibit strong peroxidase activity. When TMB was used as substrate and the product was determined at the wavelength of 450nm, it had a detection limit of 0.8 pM. The detection system has very low detection background and high selectivity of nucleic acid detection.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R440
，
本文編號：2464700
[Abstract]:G-tetraad (G-4) is a special secondary structure of nucleic acid, which is composed of oligodeoxynucleotide sequences rich in guanine base (G) through Hoogsteen bonds. In solutions containing some monovalent metal ions, G4 binds to the hemin monomer (Hemin) to form a functional oligodeoxynucleotide structure with peroxidase activity, also known as the DNA mimicase (peroxidase-mimicking DNAzyme). Using DNA mimetic enzyme as signal detection element is very portable for nucleic acid detection. However, nowadays, homogenous detection of nucleic acid based on DNA mimetic enzyme has a large detection background. This hinders the wide application of DNA mimetic enzyme in nucleic acid homogenous detection. Conjugation probe (junction probe,JP) is derived from the concept of double probe (binary probe,BP). In a system, if and only if two probes are hybridized with the target nucleic acid sequence at the same time, the hybridization signal can be generated, which makes it very specific for hybridization. And it has the potential to be used for homogenous detection of nucleic acids. Because of the large non-specific hybridization probability, the traditional conjugation probe needs complex sequence design and optimization, which increases the cost of detection. In this study, based on the previous studies, the conjugation probe based on the chain substitution technique (strand displacement) was constructed, and the G-tetraad was integrated into the portable detection of nucleic acid, and its detection efficiency was evaluated preliminarily. The thesis is divided into two chapters: in the first chapter, we integrate the split G-tetraad formation sequence into two probes. Only in the presence of transcripts containing specific splicing sites, probes can be assembled together to form a three-way conjugation conformation, thus providing a functional G-tetraad conformation. After binding with hemin monomer, the peroxidase activity of hemin can be greatly enhanced. The detection limit for target gene transcripts by colorimetric assay is 0.063 渭 M, and the system can specifically identify single base mutations in the target sequence (about 3 times the amount of mutation sequence is required for the detection system to obtain the same signal as the target sequence). The target sequence identified by the conjugation probe can be up to 46 bp, long and does not require complex sequence design steps, showing its potential for further construction of a simple and efficient nucleic acid detection system. In the second chapter, we construct a method for nucleic acid target sequence recognition and signal amplification. Nucleic acid amplification is based on chain substitution amplification (strand displacement amplification,SDA), in which two DNA probes do not assemble each other to trigger downstream strand substitution amplification under the action of inhibitory chain (inhibitor). However, in the presence of the target sequence, the probe can release the inhibitory chain through the three-way conjugation structure induced by the target sequence (see chapter I for the core principle), and then trigger the SDA reaction to amplify the C (cytosine)-rich sequence template. This leads to the formation of a series of G-rich (guanine) sequences, which combine with hemin monomers and exhibit strong peroxidase activity. When TMB was used as substrate and the product was determined at the wavelength of 450nm, it had a detection limit of 0.8 pM. The detection system has very low detection background and high selectivity of nucleic acid detection.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R440
，
本文編號：2464700
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