适于多物种的通用尾巴序列设计及通用体系的建立

Design of Universal Tailed-sequence and Establishment of the Universal System Suitable for Multiple Species

【目的】荧光毛细管电泳由于其检测通量高、分辨率高等优点,被广泛应用于个体鉴定、品种鉴定、物种鉴定等多种应用场景中。尾巴序列为荧光毛细管电泳平台的广泛应用提供了高效的解决方案。为解决已有尾巴序列无法满足多物种使用需求,本研究基于编码转译技术开发高效的通用尾巴序列(universal tailed-sequence,UTS)设计工具。基于此工具设计通用尾巴序列,构建适合多作物的通用型PCR体系和程序,提高荧光电泳通量和灵活性。【方法】基于编码转译技术开发高效的通用尾巴序列设计工具,并对3755个常用汉字进行编码转译,并设置序列GC含量、发卡结构及同源引物二聚体退火温度等筛选条件得到符合条件的UTS。使用BLAST工具对通用尾巴序列设计工具生成的UTS在多种生物基因组上进行同源性评估,并在玉米、番茄、辣椒、西瓜等作物上进行实验评估,构建物种通用型实验体系及程序。【结果】通过设计工具编码转译并筛选共得到7436833个高质量的候选UTS,占所有字组的52.74%。挑选6个UTS在20个作物基因组上的BLAST结果显示其与M13相比具有更好的特异性。通过对通用引物扩增程序进行优化,使通用引物在多个物种上的扩增成功率达到或超过95%,并具有较强的稳定性。【结论】利用编码转译技术开发通用尾巴序列,并为其搭配物种通用型扩增体系和程序,提供一种通量高、成本低的荧光电泳通用检测方法。

【Objective】Fluorescent capillary electrophoresis,for its high detection throughput and resolution,was in extensive applications in various scenarios,including individual identification,variety discrimination,and species identification.The tailed-sequence offers an efficient solution for the extensive application of the fluorescent capillary electrophoresis platforms.To address the limitation of existing tailed-sequences in meeting the demands of multi-species applications,this study developed an efficient Universal Tailed-Sequence(UTS)design tool based on encoding translation technology.Utilizing this tool,UTS were designed to construct a versatile PCR system and protocol suitable for multiple crops,thereby enhancing the throughput and flexibility of fluorescent electrophoresis.【Method】An efficient UTS design tool was designed based on encoding translation technology and 3755 commonly used Chinese characters were encoding translated with filtering criteria including GC content,hairpin,and the self-dimer annealing temperature.The BLAST tool was used to assess the homology of UTS generated by the design tool on various biological genomes.The UTS was experimentally evaluated on crops such as maize,tomatoes,peppers,and watermelons,constructing a species-universal experimental system and protocol.【Result】The design tool,through encoding translation and screening,generated a total of 7436833 high-quality candidate UTS,accounting for 52.74%of all character combinations.Six selected UTS were demonstrated better specificity across 20 crop genomes by BLAST results compared to M13.With optimization of the general primer amplification procedure,the success rate of general primer amplification across multiple species reached or exceeded 95%,showing strong stability.【Conclusion】This study utilized encoding translation technology to develop UTS and paired them with a species-universal amplification system and protocol,which may provide a high-throughput,cost-effective universal detection method for fluorescent electrophoresis.