血浆DNA直接芯片PCR扩增和微流控化学发光SNP分析系统的研究
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摘要
核酸是一类重要的生物分子,其分析一直受到人们的关注。核酸分析的发展离不开先进的分析仪器与技术,而仪器设备的微型化和集成化是一个日益明显的发展趋势,微全分析系统的提出和发展就是这一趋势的体现。血液是临床常用的检测样本,血浆DNA是血液细胞外的DNA,因为血浆DNA在疾病检测和预估方面的独特意义,近年来医学界对它的研究日益增多。本文在静态微池PCR芯片技术的基础上建立了血浆游离DNA的直接扩增方法；为了进一步对DNA进行分析,建立了微流控发光核酸分析系统,并初步用于核酸SNP位点的检测。
基于血液直接扩增和芯片PCR研究,建立了静态微池PCR芯片上血浆DNA直接扩增方法。将λDNA(扩增片段为236bp)添加于血浆中,形成游离DNA模拟样品,通过改变PCR程序,优化反应体系,建立了直接血浆DNA扩增方法。该方法成功用于血浆中DYZ-1型基因(扩增片段为149bp)的直接扩增,以凝胶电泳作为检测手段,在25μL扩增反应体系中,发现血浆量在0.1-16.71μL之间均得到目标扩增产物。并在芯片上成功地进行λDNA和DYZ-1型基因的直接扩增,扩增结果分别用凝胶电泳和芯片毛细管电泳进行了检测。
基于焦磷酸测序化学发光反应,利用磁定位固定方法在毛细管中制作了微反应器,建立了微流控发光分析系统。首先探讨了微反应器的制作,包括高梯度磁固定玻璃芯片、高梯度磁固定PDMS-ITO玻璃芯片和毛细管微反应器的制作。以毛细管作为试剂输运和发光反应的基本平台,永磁铁在毛细管两侧以相吸模式加磁固定磁珠模板,用磁屏蔽盒消除磁场对光电倍增管的可能干扰,建立微流控核酸发光分析系统。利用此系统,借助BAMPER法(结合修饰引物延伸反应的生物发光分析检测)对来自人血液的21号染色体rs8130833的SNP位点进行了检测。与基于静态发光反应的方法相比,该方法具有简单快速的特点。
DNA is an important kind of biological macromolecules, and its analysis is receiving widespread attention. Improvements of DNA analysis depend very much on method development, and miniaturization and integration are two of these trends. The appearance of micro total analysis system (Micro-TAS) is also a reflection of the development. Blood is a typical and commonly used clinical sample, and plasma DNA is cell-free DNA in the blood. Beacause of its unique significance in disease detection and diagnosis, plasma DNA ananlysis is of prime interest both in medicine and scientific research. In this thesis, a direct plasma DNA PCR amplification method was developed, and implemented on a static chip PCR system; Furthermore, a microfluidic chemiluminscence DNA analysis system was established, and a SNP typing was demonstrated with the system.
Firstly, direct plasma DNA PCR amplification on a static chip thermostat without purification procedure was investigated. The amplification conditions such as the composition of reagents and thermal programs were studied systematically with a GeneAmp PCR system with an exteriorλDNA segment (236bp) as the target, and the method was applied for DYZ-1 gene (149bp) amplification directly from plasma samples. Satisfactory results were obtained with plasma sample ranging from 0.1μL to 16.7μL within a 25μL PCR reaction system. Direct amplifications of exterior X-DNA and plasma DYZ-1 gene were successfully demonstrated by a static chip PCR as characterized by gel electrophoresis and confirmed by PCR-chip electrophoresis.
Secondly, a simple microfluidic chemilluminscence DNA analyzing system was established based on pyro-sequencing reactions. Various forms of microreactors were investigated for immobilizing the magnetic beads bearing the template, incluiding a glass HGMS (high gradient magnetic seperation) chip, a PDMS-ITO glass HGMS chip and a capillary chip. Fused silica capillary was chosen both for reagent infusion and reaction cell, and the bead templates were fixed by permanet magnets on both sides under attraction mode. A iron shielding box was used avoiding possible harm effect of magnetic to the photo multiplier. SNP typing of rs8130833 on chromosome 21 was achived taking advantage of BAMPER (Bioluminometric Assay coupled with Modified Primer Extension Reactions). Comparing to the static method where 4 enzymes were involved, this method is simpler, faster and more reagent saving.
基于血液直接扩增和芯片PCR研究,建立了静态微池PCR芯片上血浆DNA直接扩增方法。将λDNA(扩增片段为236bp)添加于血浆中,形成游离DNA模拟样品,通过改变PCR程序,优化反应体系,建立了直接血浆DNA扩增方法。该方法成功用于血浆中DYZ-1型基因(扩增片段为149bp)的直接扩增,以凝胶电泳作为检测手段,在25μL扩增反应体系中,发现血浆量在0.1-16.71μL之间均得到目标扩增产物。并在芯片上成功地进行λDNA和DYZ-1型基因的直接扩增,扩增结果分别用凝胶电泳和芯片毛细管电泳进行了检测。
基于焦磷酸测序化学发光反应,利用磁定位固定方法在毛细管中制作了微反应器,建立了微流控发光分析系统。首先探讨了微反应器的制作,包括高梯度磁固定玻璃芯片、高梯度磁固定PDMS-ITO玻璃芯片和毛细管微反应器的制作。以毛细管作为试剂输运和发光反应的基本平台,永磁铁在毛细管两侧以相吸模式加磁固定磁珠模板,用磁屏蔽盒消除磁场对光电倍增管的可能干扰,建立微流控核酸发光分析系统。利用此系统,借助BAMPER法(结合修饰引物延伸反应的生物发光分析检测)对来自人血液的21号染色体rs8130833的SNP位点进行了检测。与基于静态发光反应的方法相比,该方法具有简单快速的特点。
DNA is an important kind of biological macromolecules, and its analysis is receiving widespread attention. Improvements of DNA analysis depend very much on method development, and miniaturization and integration are two of these trends. The appearance of micro total analysis system (Micro-TAS) is also a reflection of the development. Blood is a typical and commonly used clinical sample, and plasma DNA is cell-free DNA in the blood. Beacause of its unique significance in disease detection and diagnosis, plasma DNA ananlysis is of prime interest both in medicine and scientific research. In this thesis, a direct plasma DNA PCR amplification method was developed, and implemented on a static chip PCR system; Furthermore, a microfluidic chemiluminscence DNA analysis system was established, and a SNP typing was demonstrated with the system.
Firstly, direct plasma DNA PCR amplification on a static chip thermostat without purification procedure was investigated. The amplification conditions such as the composition of reagents and thermal programs were studied systematically with a GeneAmp PCR system with an exteriorλDNA segment (236bp) as the target, and the method was applied for DYZ-1 gene (149bp) amplification directly from plasma samples. Satisfactory results were obtained with plasma sample ranging from 0.1μL to 16.7μL within a 25μL PCR reaction system. Direct amplifications of exterior X-DNA and plasma DYZ-1 gene were successfully demonstrated by a static chip PCR as characterized by gel electrophoresis and confirmed by PCR-chip electrophoresis.
Secondly, a simple microfluidic chemilluminscence DNA analyzing system was established based on pyro-sequencing reactions. Various forms of microreactors were investigated for immobilizing the magnetic beads bearing the template, incluiding a glass HGMS (high gradient magnetic seperation) chip, a PDMS-ITO glass HGMS chip and a capillary chip. Fused silica capillary was chosen both for reagent infusion and reaction cell, and the bead templates were fixed by permanet magnets on both sides under attraction mode. A iron shielding box was used avoiding possible harm effect of magnetic to the photo multiplier. SNP typing of rs8130833 on chromosome 21 was achived taking advantage of BAMPER (Bioluminometric Assay coupled with Modified Primer Extension Reactions). Comparing to the static method where 4 enzymes were involved, this method is simpler, faster and more reagent saving.
引文
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