DNA微阵列原位化学合成

doi:10.12211/2096-8280.2020-089

摘要/Abstract

摘要：

高通量、快速、低成本DNA合成是合成生物学、DNA信息存储以及DNA芯片等前沿科技领域的重要核心技术。DNA微阵列原位化学合成方法是在亚磷酸酰胺固相化学合成原理的基础上，整合了微电子学、计算科学、分子生物学、光电化学和微纳加工等学科的相关技术，近30年来得到了迅速的发展和应用。DNA微阵列原位化学合成方法根据不同的碱基分配方式可以分为原位光刻法、光敏抗蚀层合成法、光致酸法、喷印合成法、软光刻合成法、电致酸法和压印法以及以这些技术为基础衍生的各种合成方法等。本文对上述不同的DNA微阵列原位化学合成方法及其技术特点进行阐述，并对未来DNA合成方法的发展趋势进行讨论和展望。合成通量和效率方面基于CMOS芯片的电致酸DNA原位化学合成技术在未来10年内将具备较大的发展空间，通过解决芯片上微电极间氢离子串扰问题，有望实现单片TB级的DNA快速低成本合成。

关键词: 合成生物学, DNA合成, DNA原位化学合成, 微阵列, 电致酸

Abstract:

High-throughput, rapid and low-cost DNA synthesis is an important core technology in the research fields such as synthetic biology, DNA storage and DNA chips. The in-situ chemical synthesis of DNA microarrays is based on the principle of solid-phase chemical synthesis of phosphorous acid amides, and integrates the relevant technologies of microelectronics, computational science, molecular biology, photo-electrochemistry and micro-nano processing. In the past 30 years, the technology has developed rapidly. In-situ chemical synthesis methods can be grouped into photolithography, photo-acid methods, electro-acid methods, printing methods and imprinting methods, etc. based on their base allocation strategy. In this paper, we discuss the different in-situ chemical synthesis methods of DNA microarrays and their technical characteristics, as well as the potential development trends of DNA synthesis methods in the future. We believe that in terms of synthesis throughput and efficiency, the CMOS (Complementary Metal Oxide Semiconductor) chip-based in-situ chemical synthesis of electro-acid DNA has tremendous potential in the next decade. By solving the problem of hydrogen ion crosstalk between microelectrodes on the chip, it is expected that the rapid and low-cost synthesis of TB-level DNA can be accomplished on a single chip. As shown in the schematic diagram, the voltage of different areas of the DNA synthesis chip is controlled by computer to selectively deprotect the bases at different sites to achieve high-throughput parallel synthesis of different sequences of DNA. However, the bottleneck comes from the crosstalk between micro-electrodes due to acidic ion diffusion inside CMOS chip, which results in the limitation of synthetic capacity at the hundreds-of-megabyte level. After studying the behavior of hydrogen ion transport at the micrometer scale, the redesigning of CMOS chip with new materials and structures is suggested, aiming to suppress the ion diffusion between micro-electrodes and optimize the structure of microelectrodes and microfluidic channels of the chip and device. It is possible that the synthetic capacity of single chip could reach terabyte-level for CMOS-based in situ parallel DNA chemical synthesis with electro-acidified deprotection in the future, which will significantly accelerate the practical applications of synthetic biology and related technologies.

Key words: synthetic biology, DNA synthesis, DNA microarray in situ chemical synthesis, microarray, electro-acidification

中图分类号:

闫汉, 肖鹏峰, 刘全俊, 陆祖宏. DNA微阵列原位化学合成[J]. 合成生物学, 2021, 2(3): 354-370.

Han YAN, Pengfeng XIAO, Quanjun LIU, Zuhong LU. In situ chemical synthesis of DNA microarrays[J]. Synthetic Biology Journal, 2021, 2(3): 354-370.

图/表 11

参考文献 111

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方法	技术特征	优点	缺点
原位光刻法	光通过掩模（或者虚拟掩模）照射特定的阵点，5'端保护基团发生光化学反应被直接脱除，被活化的5'-羟基可以进行偶联、封闭、氧化反应	光刻的分辨率高。可合成10⁶DNA序列/cm²，合成长度60 nt（已商业化）	与传统亚磷酸酰胺法单体试剂不兼容，光干扰影响偶联效率
光敏抗蚀层合成法	光通过掩模（或者虚拟掩模）照射特定的阵点。将光敏抗蚀层清除，采用常用的酸脱保护剂，将暴露区域5′-羟基保护基团脱除，被活化的5′端羟基可以进行偶联、封闭、氧化反应	光刻分辨率高，与传统亚磷酸酰胺法单体试剂兼容。可合成10⁶DNA序列/cm²，合成长度20 nt	涂胶、显影、去胶等复杂的光刻工艺，极易污染合成芯片，同时影响被保护寡核苷酸的稳定性
光致酸法	光通过掩模（或者虚拟掩模）照射特定的阵点，使阵点上物质发生光化学反应，产生的酸将5′端保护基团脱除，被活化的5′端羟基可以进行偶联、封闭、氧化反应	光刻分辨率高，单位面积上阵点数目多。可合成10⁶DNA序列/cm²，合成长度120 nt	与传统亚磷酸酰胺法脱保护试剂不兼容，需选择合适的光致酸试剂，需防止酸扩散污染
喷印合成法	在特定阵点上喷印单体进行偶联	与传统亚磷酸酰胺法兼容，合成效率高。可合成10³DNA序列/cm²，合成长度120 nt	喷头的机械定位精度要求高，喷印的位置无法做到完全重叠，不适合高密度DNA阵列合成
电致酸法	在特定阵点的电极通电，使阵点周围物质发生电化学反应，产生的酸将5′端保护基团脱除，被活化的5′端羟基可以进行偶联、封闭、氧化反应	半导体制造技术，合成区域定位准确。可合成10⁵DNA序列/cm²，合成长度160 nt（已商业化）	与传统亚磷酸酰胺法脱保护试剂不兼容，需选择合适的电致酸试剂，需防止酸扩散污染
压印法	在特定阵点上压印单体进行偶联	与传统亚磷酸酰胺法兼容，合成效率高。可合成6×10⁴DNA序列/cm²，合成长度60 nt（已商业化）	压印机械定位精度要求高，两个接触面需高度平行

方法	技术特征	优点	缺点
原位光刻法	光通过掩模（或者虚拟掩模）照射特定的阵点，5'端保护基团发生光化学反应被直接脱除，被活化的5'-羟基可以进行偶联、封闭、氧化反应	光刻的分辨率高。可合成10⁶DNA序列/cm²，合成长度60 nt（已商业化）	与传统亚磷酸酰胺法单体试剂不兼容，光干扰影响偶联效率
光敏抗蚀层合成法	光通过掩模（或者虚拟掩模）照射特定的阵点。将光敏抗蚀层清除，采用常用的酸脱保护剂，将暴露区域5′-羟基保护基团脱除，被活化的5′端羟基可以进行偶联、封闭、氧化反应	光刻分辨率高，与传统亚磷酸酰胺法单体试剂兼容。可合成10⁶DNA序列/cm²，合成长度20 nt	涂胶、显影、去胶等复杂的光刻工艺，极易污染合成芯片，同时影响被保护寡核苷酸的稳定性
光致酸法	光通过掩模（或者虚拟掩模）照射特定的阵点，使阵点上物质发生光化学反应，产生的酸将5′端保护基团脱除，被活化的5′端羟基可以进行偶联、封闭、氧化反应	光刻分辨率高，单位面积上阵点数目多。可合成10⁶DNA序列/cm²，合成长度120 nt	与传统亚磷酸酰胺法脱保护试剂不兼容，需选择合适的光致酸试剂，需防止酸扩散污染
喷印合成法	在特定阵点上喷印单体进行偶联	与传统亚磷酸酰胺法兼容，合成效率高。可合成10³DNA序列/cm²，合成长度120 nt	喷头的机械定位精度要求高，喷印的位置无法做到完全重叠，不适合高密度DNA阵列合成
电致酸法	在特定阵点的电极通电，使阵点周围物质发生电化学反应，产生的酸将5′端保护基团脱除，被活化的5′端羟基可以进行偶联、封闭、氧化反应	半导体制造技术，合成区域定位准确。可合成10⁵DNA序列/cm²，合成长度160 nt（已商业化）	与传统亚磷酸酰胺法脱保护试剂不兼容，需选择合适的电致酸试剂，需防止酸扩散污染
压印法	在特定阵点上压印单体进行偶联	与传统亚磷酸酰胺法兼容，合成效率高。可合成6×10⁴DNA序列/cm²，合成长度60 nt（已商业化）	压印机械定位精度要求高，两个接触面需高度平行

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