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

doi:10.13560/j.cnki.biotech.bull.1985.2023-0967

Abstract

Abstract:

【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 3 755 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 7 436 833 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.

Key words: PCR, fluorescence electrophoresis, universal primer, primer design

SUN Bo, WANG Rui, HUO Yong-xue, GE Jian-rong, KUANG Meng, WANG Feng-ge. Design of Universal Tailed-sequence and Establishment of the Universal System Suitable for Multiple Species[J]. Biotechnology Bulletin, 2024, 40(5): 94-102.

Figures/Tables 10

References 22

[1]	谭智广. 低功耗聚合酶链式反应温度控制系统的研制[D]. 大连: 大连理工大学, 2023.
	Tan GZ. Development of low power consumption temperature control system of PCR[D]. Dalian: Dalian University of Technology, 2023.
[2]	Zhu HL, Zhang HQ, Xu Y, et al. PCR past, present and future[J]. BioTechniques, 2020, 69(4): 317-325.
[3]	郑戈文. 农作物种子检测中常用电泳方法的比较分析[J]. 中国种业, 2018(11): 35-37.
	Zheng GW. Comparative analysis of electrophoresis methods commonly used in crop seed detection[J]. China Seed Ind, 2018(11): 35-37.
[4]	王爱娜, 王灏, 李殿荣, 等. 琼脂糖与聚丙烯酰胺凝胶电泳在SSR鉴定杂交油菜种子纯度中的比较[J]. 西北农业学报, 2012, 21(8): 101-106.
	Wang AN, Wang H, Li DR, et al. Comparison of agarose and polyacrylamide gel electrophoresis for SSR in purity identification of qinyou 33 hybrid rape seed[J]. Acta Agric Boreali Occidentalis Sin, 2012, 21(8): 101-106.
[5]	王竹林, 杨睿, 刘联正, 等. 聚丙烯酰胺凝胶电泳银染的影响因素[J]. 实验室研究与探索, 2011, 30(9): 15-17, 113.
	Wang ZL, Yang R, Liu LZ, et al. Factors of silver staining of polyacrylamide gel electrophoresis[J]. Res Explor Lab, 2011, 30(9): 15-17, 113.
[6]	冯琬淇, 张福顺, 刘乃新. 毛细管与聚丙烯酰胺凝胶电泳检测甜菜SSR位点的比较研究[J]. 中国农学通报, 2022, 38(36): 34-41. doi: 10.11924/j.issn.1000-6850.casb2022-0412
	Feng WQ, Zhang FS, Liu NX. SSR polymorphism in sugar beet: detection by polyacrylamide gel electrophoresis and capillary electrophoresis[J]. Chin Agric Sci Bull, 2022, 38(36): 34-41. doi: 10.11924/j.issn.1000-6850.casb2022-0412
[7]	刘文彬, 许理文, 王凤格, 等. 基于两种荧光毛细管电泳平台筛选评估玉米新型SSR引物[J]. 玉米科学, 2017, 25(2): 24-30.
	Liu WB, Xu LW, Wang FG, et al. Evaluating and screening new maize SSR primer based on two kinds of fluorescent capillary eelectrophoresis platform[J]. J Maize Sci, 2017, 25(2): 24-30.
[8]	管俊娇, 杨晓洪, 张鹏, 等. 基于荧光检测技术的青稞品种鉴定方法的建立[J]. 生物技术通报, 2021, 37(11): 293-302. doi: 10.13560/j.cnki.biotech.bull.1985.2020-1494
	Guan JJ, Yang XH, Zhang P, et al. Establishment of a method identifying the varieties of hulless barley based on fluorescence detection technology[J]. Biotechnol Bull, 2021, 37(11): 293-302.
[9]	方梅, 胡波, 侯媛媛, 等. 毛细管电泳在基因研究中的应用[J]. 生物技术通报, 2006(2): 60-66.
	Fang M, Hu B, Hou YY, et al. The application of capillary electrophoresis to gene analysis[J]. Biotechnol Bull, 2006(2): 60-66.
[10]	李会勇, 王天宇, 黎裕, 等. TP-M13自动荧光检测法在高粱SSR基因型鉴定中的应用[J]. 植物遗传资源学报, 2005, 6(1): 68-70.
	Li HY, Wang TY, Li Y, et al. Application of the TP-M13 automated fluorescent-lablled system of SSR genotyping in sorghum[J]. J Plant Genet Resour, 2005, 6(1): 68-70.
[11]	Schuelke M. An economic method for the fluorescent labeling of PCR fragments[J]. Nat Biotechnol, 2000, 18(2): 233-234. doi: 10.1038/72708 pmid: 10657137
[12]	匡猛, 王凤格, 赵久然, 等. 玉米SSR引物尾巴序列的设计方法[J]. 农业生物技术学报, 2007, 15(6): 1072-1073.
	Kuang M, Wang FG, Zhao JR, et al. A method of designing tail sequence for maize SSR primers[J]. J Agric Biotechnol, 2007, 15(6): 1072-1073.
[13]	曾斌, 马鑫鑫, 余镇藩, 等. 新疆野扁桃的TP-M13-SSR荧光标记引物的筛选[J]. 中国农学通报, 2019, 35(1): 45-49. doi: 10.11924/j.issn.1000-6850.casb18070067
	Zeng B, Ma XX, Yu ZF, et al. Wild almond(Prunus tenella batsch.) in Xinjiang: screening of TP-M13-SSR primers in capillary fluorescent electrophoresis[J]. Chin Agric Sci Bull, 2019, 35(1): 45-49.
[14]	赵紫薇, 任洁, 刘亚维, 等. 一种适用于玉米胚乳DNA提取的新方法[J]. 分子植物育种, 2023: 1-10.
	Zhao ZW, Ren J, Liu YW, et al. A New Method for DNA Extraction from Maize Endosperm[J]. Molecular Plant Breeding, 2023: 1-10.
[15]	国家市场监督管理总局, 国家标准化管理委员会. 主要农作物品种真实性和纯度SSR分子标记检测玉米: GB/T 39914—2021[S]. 北京: 中国标准出版社, 2021.
	State Administration of Market Supervision and administration, Standardization Administration of the People's Republic of China. Variety genuineness and purity testing of main crops with SSR markers—Maize: GB/T 39914—2021[S]. Beijing: Standards Press of China, 2021.
[16]	中华人民共和国农业部. 番茄品种鉴定技术规程 Indel分子标记法: NY/T 2471—2013[S]. 北京: 中国农业出版社, 2014.
	Ministry of Agriculture of the People's Republic of China. Identification of tomato varieties—Indel marker method: NY/T 2471—2013[S]. Beijing: China Agriculture Press, 2014.
[17]	中华人民共和国农业部. 西瓜品种鉴定技术规程 SSR分子标记法: NY/T 2472—2013[S]. 北京: 中国农业出版社, 2014.
	Ministry of Agriculture of the People's Republic of China. Identification of watermelon varieties—SSR marker method: NY/T 2472—2013[S]. Beijing: China Agriculture Press, 2014.
[18]	中华人民共和国农业部. 辣椒品种鉴定技术规程 SSR分子标记法: NY/T 2475—2013[S]. 北京: 中国农业出版社, 2014.
	Ministry of Agriculture of the People's Republic of China. Identification of pepper varieties—SSR marker method: NY/T 2475—2013[S]. Beijing: China Agriculture Press, 2014.
[19]	刘志斋, 王天宇, 黎裕. TP-M13-SSR技术及其在玉米遗传多样性研究中的应用[J]. 玉米科学, 2007, 15(6): 10-15, 31.
	Liu ZZ, Wang TY, Li Y. TP-M13-SSR technique and its applications in the analysis of genetic diversity in maize[J]. J Maize Sci, 2007, 15(6): 10-15, 31.
[20]	李小娟, 刘文雅, 何彦峰, 等. 葫芦藓叶绿体基因组密码子使用模式分析[J]. 分子植物育种, 2023: 1-15.
	Li XJ, Liu WY, He YF, et al. Analysis of codon usage patterns in chloroplast genomes of funaria hygrometrica[J]. Molecular Plant Breeding, 2023: 1-15.
[21]	仇学文, 李丹, 甘玉迪, 等. 豇豆叶绿体基因组密码子使用偏好性分析[J]. 核农学报, 2023, 37(6): 1118-1129. doi: 10.11869/j.issn.1000-8551.2023.06.1118
	Qiu XW, Li D, Gan YD, et al. Analysis of codon usage bias in the cowpea chloroplast genome[J]. J Nucl Agric Sci, 2023, 37(6): 1118-1129. doi: 10.11869/j.issn.1000-8551.2023.06.1118
[22]	雷佳欣, 张丽娟, 高丹丹, 等. 文冠果基因组密码子偏好性分析[J]. 西北林学院学报, 2023, 38(4): 104-110.
	Lei JX, Zhang LJ, Gao DD, et al. Codon usage bias of Xanthoceras sorbifolium genome[J]. J Northwest For Univ, 2023, 38(4): 104-110.

反应组分 Reaction component	荧光引物扩增体系 Fluorescent primer amplification system	TP-M13-SSR扩增体系 TP-M13-SSR amplification system	UTS通用扩增体系 UTS universal amplification system
ddH₂O	/	/	/
10×Buffer	1×	1×	1×
MgCl₂	2.5 mmol/L	2.5 mmol/L	2.5 mmol/L
dNTPs	0.15 mmol/L each	0.15 mmol/L each	0.15 mmol/L each
Taq 酶 Taq enzyme	1.0 U	1.0 U	1.0 U
上游引物 Forward primer	0.25 μmol/L	0.015 μmol/L	0.03 μmol/L
下游引物 Reverse primer	0.25 μmol/L	0.1 μmol/L	0.1 μmol/L
通用尾巴序列 Universal tailed-sequence	/	0.1 μmol/L	0.1 μmol/L
模板DNA Template DNA	5 ng/μL	5 ng/μL	5 ng/μL

反应组分 Reaction component	荧光引物扩增体系 Fluorescent primer amplification system	TP-M13-SSR扩增体系 TP-M13-SSR amplification system	UTS通用扩增体系 UTS universal amplification system
ddH₂O	/	/	/
10×Buffer	1×	1×	1×
MgCl₂	2.5 mmol/L	2.5 mmol/L	2.5 mmol/L
dNTPs	0.15 mmol/L each	0.15 mmol/L each	0.15 mmol/L each
Taq 酶 Taq enzyme	1.0 U	1.0 U	1.0 U
上游引物 Forward primer	0.25 μmol/L	0.015 μmol/L	0.03 μmol/L
下游引物 Reverse primer	0.25 μmol/L	0.1 μmol/L	0.1 μmol/L
通用尾巴序列 Universal tailed-sequence	/	0.1 μmol/L	0.1 μmol/L
模板DNA Template DNA	5 ng/μL	5 ng/μL	5 ng/μL

通用尾巴序列类别 UTS category	扩增程序 Amplification program
UTS通用扩增程序 UTS universal amplification program	95℃，5 min； 95℃，40 s，55℃，35 s，72℃，45 s，35 cycle； 72℃，10 min；4℃ store
TP-M13-SSR扩增程序 TP-M13-SSR amplification program	95℃，5 min； 95℃，40 s，Primer annealing temperature，35 s，72℃，45 s，8 cycle； 95℃，40 s，M13 Tailed-sequence annealing temperature，35 s，72℃，45 s，30 cycle； 72℃，10 min；4℃

通用尾巴序列类别 UTS category	扩增程序 Amplification program
UTS通用扩增程序 UTS universal amplification program	95℃，5 min； 95℃，40 s，55℃，35 s，72℃，45 s，35 cycle； 72℃，10 min；4℃ store
TP-M13-SSR扩增程序 TP-M13-SSR amplification program	95℃，5 min； 95℃，40 s，Primer annealing temperature，35 s，72℃，45 s，8 cycle； 95℃，40 s，M13 Tailed-sequence annealing temperature，35 s，72℃，45 s，30 cycle； 72℃，10 min；4℃

UTS编号UTS code	汉字字组 Chinese character	编码后序列 Sequence after encoding（5'-3'）	序列GC含量 GC content of sequence/%	发卡结构T_m Hairpin T_mvalue/℃	同源二聚体T_m值 Self-dimer T_m value/℃
UTS01	孙擘（Sun Bo）	GACCAAGCACACGACAACTGACAT	50.0	/	-29.7
UTS02	电泳（Dian Yong）	GACGACCTAGCCGACAAGTGAGTG	58.3	/	9.3
UTS03	荧光（Ying Guang）	GAATATGCAACGGACCATCCATAC	45.8	35.9	-25.8
UTS04	基因（Ji Yin）	GACCACGGAGAAGACCACAGAATT	50.0	/	-19.6
UTS05	分子（Fen Zi）	GACCATATATCAGACCAAGCACTT	41.7	/	-33.0
UTS06	玉米（Yu Mi）	GACGATGAATACGACGAGTCAGTG	50.0	34.7	-0.4