基因点突变检测新方法及新型界面设计的免疫传感器研究
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摘要
人类的许多遗传性疾病都是由于基因的变异引起的,其中尤以单碱基的突变,即单碱基多态性最为普遍。实现对这些基因单碱基突变的早期、准确、简便、快速的确认,对于人类相关疾病的发病机理研究及早期治疗具有非常重要的意义。目前,有关单碱基突变的检测方法已有许多报道。而这些传统的方法普遍操作繁琐费时、不易定量、仪器昂贵、且对工作人员要求较高的专业技能,因此仅能在少数实验室应用。开发一些简便、价廉、精确且易于临床推广的方法是一件很有价值的工作。此外,由于电化学生物传感器具有制造简单、灵敏度高、价格低廉而被广泛研究、并已在生物检测中逐步得到了应用。然而,有效的生物活性组分的固定方法是构建性能优良生物传感器的关键,如何将生物活性组分有效地固定在电极表面的固定化方法,以及传感器的重现性和重复使用性等方面存在的问题,严重阻碍了生物传感器的进一步发展和应用。基于当前基因单碱基突变检测技术和生物传感技术敏感膜构建中存在的问题,本研究论文发展了一系列新的基因单碱基突变检测技术及新型界面设计的生物传感器,实现了对目标物的高灵敏测定。主要内容如下:
(1)利用核酸标记金纳米颗粒凝集变色的光学特性,提出了一种基于DNA连接酶反应和纳米金聚集特性的比色检测方法,用于单碱基突变的检测(第2章)。通过将高忠实性DNA连接酶的等位特异连接特性与纳米金聚集体系相结合,发展了一种简便且不需要精确温度控制的比色检测方法,可以用于单碱基突变的直接检测。此外,连接反应能在较高的温度下进行,因而减少了因非相关链与探针标记纳米金的非特异作用而引起的背景干扰。该方法可以分为三步来完成:首先是杂交反应,使两个金标探针与DNA目标链杂交形成双链结构;随后的连接反应中,使完全匹配的连接、错配的不连接;最后由热处理步骤来分析探针的连接情况。当加热反应混合液,使形成的DNA双链熔链时,对于完全匹配的情况,溶液的颜色不会恢复到红色。而对于错配的情况,由于聚集金纳米颗粒的重新解离,溶液的颜色又回复到红色。我们用该方法对结肠癌k-ras基因的第12位密码子的突变情况进行了检测,使突变型和野生型得到了很好的区分。此后,我们又将这一检测体系发展用于同种溶液中多目标链的同时检测(第3章)。简言之,长链DNA标记的纳米金在较高的温度下首先发生杂交反应,接着在较低的温度下,金标的短链DNA发生杂交反应。然后,在连接反应中,完全匹配的进行连接,而有错配的不能发生连接。最后,逐步升温进行分析,当温度达到某一形成的双链的熔链温度时,对于完全匹配的情况,溶液的紫外光谱无改变。而对于有错配的情况,由于纳米金聚集体的解离,溶液的光谱发生改变。使用该方法,对β地中海贫血基
Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. Among these changes, the point mutation, i.e. single nucleotide polymorphisms (SNPs), are the most abundant form of genetic variation. Achieving early, accurate, simple and rapid identification to these single-base mutations is of particular importance for the pathogeny and early therapy of corresponding diseases. Up to now, many techniques have been developed for SNP detection. However, these conventional procedures are generally time-consuming, lowly quantitative, complicated, and necessary for expensive instrumentation and technical skills, which are applied to date in very few laboratories. Accordingly, exploring some simple, cost-effective, accurate and easy to be clinically popularized detection methods for SNPs is still of considerable interest. In additional, the electrochemical biosensors have been widely used for the assay of biological analyte due to the advantages of this approach including their simple-design, high-sensitivity and low-cost. However, the method of immobilization, the reproducibility and the reusability still remain to be solved in the design and applications of these sensors. Among these problems presented, the key step for the fabrication of biosensors with excellent property is the immobilization of bio-species on the surface of transducers.
Focused on these said topics in the detection techniques for SNPs and the formation of the biosensing interface, several new detection methods for SNPs and several new procedures for immobilizing bio-species to construct biosensor have been developed in the presented paper and described as follows:
(1) Using the distance-dependent optical properties of aggregated Au nanoparticles functionalized with oligonucleotides, we first reported a novel colorimetric detection system for SNPs through the gold nanoparticle assembly and the ligase reaction (in Chapter 2). By incorporating the high fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold
(1)利用核酸标记金纳米颗粒凝集变色的光学特性,提出了一种基于DNA连接酶反应和纳米金聚集特性的比色检测方法,用于单碱基突变的检测(第2章)。通过将高忠实性DNA连接酶的等位特异连接特性与纳米金聚集体系相结合,发展了一种简便且不需要精确温度控制的比色检测方法,可以用于单碱基突变的直接检测。此外,连接反应能在较高的温度下进行,因而减少了因非相关链与探针标记纳米金的非特异作用而引起的背景干扰。该方法可以分为三步来完成:首先是杂交反应,使两个金标探针与DNA目标链杂交形成双链结构;随后的连接反应中,使完全匹配的连接、错配的不连接;最后由热处理步骤来分析探针的连接情况。当加热反应混合液,使形成的DNA双链熔链时,对于完全匹配的情况,溶液的颜色不会恢复到红色。而对于错配的情况,由于聚集金纳米颗粒的重新解离,溶液的颜色又回复到红色。我们用该方法对结肠癌k-ras基因的第12位密码子的突变情况进行了检测,使突变型和野生型得到了很好的区分。此后,我们又将这一检测体系发展用于同种溶液中多目标链的同时检测(第3章)。简言之,长链DNA标记的纳米金在较高的温度下首先发生杂交反应,接着在较低的温度下,金标的短链DNA发生杂交反应。然后,在连接反应中,完全匹配的进行连接,而有错配的不能发生连接。最后,逐步升温进行分析,当温度达到某一形成的双链的熔链温度时,对于完全匹配的情况,溶液的紫外光谱无改变。而对于有错配的情况,由于纳米金聚集体的解离,溶液的光谱发生改变。使用该方法,对β地中海贫血基
Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. Among these changes, the point mutation, i.e. single nucleotide polymorphisms (SNPs), are the most abundant form of genetic variation. Achieving early, accurate, simple and rapid identification to these single-base mutations is of particular importance for the pathogeny and early therapy of corresponding diseases. Up to now, many techniques have been developed for SNP detection. However, these conventional procedures are generally time-consuming, lowly quantitative, complicated, and necessary for expensive instrumentation and technical skills, which are applied to date in very few laboratories. Accordingly, exploring some simple, cost-effective, accurate and easy to be clinically popularized detection methods for SNPs is still of considerable interest. In additional, the electrochemical biosensors have been widely used for the assay of biological analyte due to the advantages of this approach including their simple-design, high-sensitivity and low-cost. However, the method of immobilization, the reproducibility and the reusability still remain to be solved in the design and applications of these sensors. Among these problems presented, the key step for the fabrication of biosensors with excellent property is the immobilization of bio-species on the surface of transducers.
Focused on these said topics in the detection techniques for SNPs and the formation of the biosensing interface, several new detection methods for SNPs and several new procedures for immobilizing bio-species to construct biosensor have been developed in the presented paper and described as follows:
(1) Using the distance-dependent optical properties of aggregated Au nanoparticles functionalized with oligonucleotides, we first reported a novel colorimetric detection system for SNPs through the gold nanoparticle assembly and the ligase reaction (in Chapter 2). By incorporating the high fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold
引文
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