一种用于PCR的番茄DNA快速粗提方法

doi:10.13560/j.cnki.biotech.bull.1985.2021-1123

摘要/Abstract

摘要：

在对植物进行分子生物学研究时需要提取植物基因组DNA并对其进行PCR克隆,目前实验室常用CTAB法提取植物DNA,但其步骤相对繁琐,需要用有机溶剂反复抽提,在样本较多时,耗时较长。NaOH可以提取多种物种DNA,本研究优化了实验室条件下更加快速、经济、安全、高效的提取植物DNA的方式,主要包括以下步骤：利用液氮快速粉碎植物样品;采用碱裂解（0.5 mol/L NaOH）的方式一步提取DNA;提取的DNA利用TE缓冲液进行中和;以中和的粗提DNA作为模板进行PCR反应。本研究利用简单的DNA提取流程并结合PCR检测方式,完成了番茄转化子的快速初步鉴定。同时还证明了该NaOH法可用于番茄不同组织的DNA提取。本研究中整个操作过程步骤简单,耗时短,可在短时间内完成大量植物样品的DNA提取,无需使用氯仿、异丙醇等有毒物质,完成提取时间约为CTAB提取法的1/5,而且提取的不同组织DNA完整性较好,质量可观,满足常规的PCR检测,可用于基因克隆及转化子筛选鉴定,具有一定的利用价值和应用前景。

关键词: 番茄, NaOH, DNA提取, PCR, 转化子鉴定

Abstract:

It is necessary to extract plant genomic DNA and perform PCR cloning when conducting the studies of molecular biology on plants. Currently,the CTAB（Cetyltrimethyl Ammonium Bromide）is commonly used to extract plant DNA in laboratories,but the steps are relatively cumbersome and require multiple organic solvents. Specifically,it takes a long time when there are many samples. The DNAs from multi-species were extracted by NaOH,and this study introduces a method of extracting plant genome DNA,which is faster,economical,safer and more efficient. The major steps include the followings：Quickly grinding the plant tissues using liquid nitrogen;quickly extracting DNA by alkaline lysis（0.5 mol/L NaOH）;neutralizing the extracted DNA sample with TE buffer;and performing PCR with neutralized DNA as template. Using this simple DNA extraction method and combining PCR detection,the rapid preliminary identification of transgenic S. lycopersicum was completed. Furthermore,it was confirmed that this NaOH method could be used for extracting DNA from the different tissues of S. lycopersicum. The entire extraction process was simple and of short-time. Thereby,DNA extraction for a large number of plant samples could be completed in a short time without using toxic regents such as chloroform and isopropanol etc. The completion time of DNA extraction is about 1/5 of the time while using CTAB method. In addition,the extracted DNA from different tissues are of good integrity and high quality,which meet regular PCR detection,which can be used for gene cloning and for transgenic tomato screening. This method demonstrates potential utilization value and prospects.

Key words: Solanum lycopersicum, NaOH, DNA extraction, PCR, identification of transformant

申恒, 刘思慧, 李跃, 李敬涛, 梁文星. 一种用于PCR的番茄DNA快速粗提方法[J]. 生物技术通报, 2022, 38(6): 74-80.

SHEN Heng, LIU Si-hui, LI yue, LI Jing-tao, LIANG Wen-xing. Rapid Crude Extraction of Genomic DNA from Solanum lycopersicum for PCR[J]. Biotechnology Bulletin, 2022, 38(6): 74-80.

图/表 9

表1 NaOH法及CTAB法提取番茄DNA的比较

Table 1 Comparison of NaOH and CTAB method for extracting S. lycopersicum DNA

DNA提取方法 Methods for DNA extraction	仪器 Instrument	危险试剂 Hazardous reagent	累计时长Total time/h
NaOH	组织研磨仪	NaOH	约0.5
CTAB	组织研磨仪,水浴锅	CTAB,氯仿,异戊醇,乙醇	约2.5

图1 不同NaOH浓度提取番茄基因组的核酸定量检测

Fig. 1 Quantitative detection of extracted genomic DNA from S. lycopersicum with different NaOH concen-trations

图2 不同NaOH处理时间对番茄基因组PCR检测

Fig. 2 PCR detection of S. lycopersicum genome with diff-erent NaOH treatment time

图3 不同方法提取番茄基因组的核酸定量检测

Fig. 3 Quantitative detection of extracted genomic DNA from S. lycopersicum with different methods

图4 不同方法提取基因组的PCR检测

Fig. 4 PCR detection of extracted genome by different methods

图5 利用NaOH方法提取番茄转化子叶片基因组并进行PCR检测

Fig. 5 PCR detection of the extracted leaf genome of S. lycopersicum transformants using NaOH method

图6 番茄转化子的PCR检测及转化子测序验证 A：利用NaOH和CTAB法进行番茄转化子PCR检测;B：1号转化子的PCR产物测序分析

Fig. 6 PCR detection and sequencing verification of S. lycopersicum transformants A：PCR detection of the extracted genome of S. lycopersicum transformants using NaOH and CTAB methods. B：Sequencing analysis of PCR products from S. lycopersicum transformant 1

图7 番茄幼苗不同组织DNA提取及PCR检测 A：番茄不同部位的组织;B：不同组织样品DNA的PCR检测结果,检测基因为SlActin

Fig. 7 DNA extraction and PCR detection from the differ-ent tissues of S. lycopersicum seedlings A：Different S. lycopersicum tissues. B：PCR results of DNA from different tissue samples. The detected gene is SlActin

图8 番茄组织DNA提取及PCR检测简单流程

Fig. 8 Schematic diagram illustrating the process of DNA extraction and PCR detection from S. lycopersicum tissues

参考文献 21

[1]	田永强, 苏敏, 赵洪林, 等. 微波法快速提取丝状真菌基因组DNA[J]. 中国抗生素杂志, 2008, 33(11):703-705.
	Tian YQ, Su M, Zhao HL, et al. Rapid extraction of genomic DNA from filamentous fungi by microwave[J]. Chin J Antibiot, 2008, 33(11):703-705.
[2]	刘秀丽, 吕红, 范小峰. 两种茄科植物基因组提取方法的比较[J]. 陇东学院学报, 2017, 28(1):53-56.
	Liu XL, Lv H, Fan XF. A comparison on the methods for genome extraction on two plant species of Solanaceae[J]. J Longdong Univ, 2017, 28(1):53-56.
[3]	王军, 杨传平, 刘桂丰. 木本植物基因组DNA提取及鉴定[J]. 植物研究, 2006, 26(5):589-594.
	Wang J, Yang CP, Liu GF. Extraction of genomic DNA from woody plants and it’s identification[J]. Bull Bot Res, 2006, 26(5):589-594.
[4]	许峰, 刘宇, 王守现, 等. 一种适于PCR反应的快速提取食用菌基因组DNA的方法[J]. 生物技术, 2011, 21(1):43-44.
	Xu F, Liu Y, Wang SX, et al. A rapid method for extraction of genomic DNA of mushrooms for PCR analysis[J]. Biotechnology, 2011, 21(1):43-44.
[5]	柴建芳, 刘旭, 贾继增. 一种适于PCR扩增的小麦基因组DNA快速提取法[J]. 植物遗传资源学报, 2006, 7(2):246-248.
	Chai JF, Liu X, Jia JZ. A rapid isolation method of wheat DNA suitable for PCR analysis[J]. J Plant Genet Resour, 2006, 7(2):246-248.
[6]	陈吉良, 黄小龙, 吴安迪, 等. 一种快速高效提取病原真菌DNA作为PCR模板的方法[J]. 菌物学报, 2011, 30(1):147-149.
	Chen JL, Huang XL, Wu AD, et al. An efficient extraction method of pathogenic fungus DNA for PCR[J]. Mycosystema, 2011, 30(1):147-149.
[7]	Zou Y, Mason MG, Wang Y, et al. Nucleic acid purification from plants, animals and microbes in under 30 seconds[J]. PLoS Biol, 2017, 15(11):e2003916.
[8]	Govindarajan AV, Ramachandran S, Vigil GD, et al. A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world;an example of microfluidic origami[J]. Lab a Chip, 2012, 12(1):174-181. doi: 10.1039/C1LC20622B URL
[9]	Jangam SR, Yamada DH, McFall SM, et al. Rapid, point-of-care extraction of human immunodeficiency virus type 1 proviral DNA from whole blood for detection by real-time PCR[J]. J Clin Microbiol, 2009, 47(8):2363-2368. doi: 10.1128/JCM.r00092-09 pmid: 19644129
[10]	Liu C, Geva E, Mauk M, et al. An isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases[J]. Analyst, 2011, 136(10):2069-2076. doi: 10.1039/c1an00007a URL
[11]	McFall SM, Wagner RL, Jangam SR, et al. A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR[J]. J Virol Methods, 2015, 214:37-42. doi: 10.1016/j.jviromet.2015.01.005 pmid: 25681524
[12]	Rodriguez NM, Wong WS, Liu L, et al. A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples[J]. Lab Chip, 2016, 16(4):753-763. doi: 10.1039/c5lc01392e pmid: 26785636
[13]	Kim J, Mauk M, Chen DF, et al. A PCR reactor with an integrated alumina membrane for nucleic acid isolation[J]. Anal, 2010, 135(9):2408-2414. doi: 10.1039/c0an00288g URL
[14]	Oblath EA, Henley WH, Alarie JP, et al. A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva[J]. Lab Chip, 2013, 13(7):1325-1332. doi: 10.1039/c3lc40961a pmid: 23370016
[15]	Shi SR, Datar R, Liu C, et al. DNA extraction from archival formalin-fixed, paraffin-embedded tissues:heat-induced retrieval in alkaline solution[J]. Histochem Cell Biol, 2004, 122(3):211-218. doi: 10.1007/s00418-004-0693-x URL
[16]	Werner O, Ros RM, Guerra J. Direct amplification and NaOH extraction:two rapid and simple methods for preparing bryophyte DNA for polymerase chain reaction(PCR)[J]. J Bryol, 2002, 24(2):127-131. doi: 10.1179/037366802125000980 URL
[17]	Huang SW, Chan JPW, Shia WY, et al. The utilization of a commercial soil nucleic acid extraction kit and PCR for the detection of Clostridium tetanus and Clostridium chauvoei on farms after flooding in Taiwan[J]. The Journal Veterinary Medical Science, 2013, 75(4):489-495. doi: 10.1292/jvms.12-0271 URL
[18]	陈昆松, 李方, 徐昌杰, 等. 改良CTAB法用于多年生植物组织基因组DNA的大量提取[J]. 遗传, 2004, 26(4):529-531.
	Chen KS, Li F, Xu CJ, et al. An efficient macro-method of genomic DNA isolation from Actinidia chinensis leaves[J]. Hered:Beijing, 2004, 26(4):529-531.
[19]	李葱葱, 刘娜, 李飞武, 等. 一种快速提取玉米基因组DNA的方法[J]. 玉米科学, 2011, 19(1):52-55.
	Li CC, Liu N, Li FW, et al. Method for rapid abstraction of maize genome DNA[J]. J Maize Sci, 2011, 19(1):52-55.
[20]	Bucher BJ, Muchaamba G, Kamber T, et al. LAMP assay for the detection of Echinococcus multilocularis eggs isolated from canine faeces by a cost-effective NaOH-based DNA extraction method[J]. Pathogens, 2021, 10(7):847. doi: 10.3390/pathogens10070847 URL
[21]	Karakousis A, Tan L, Ellis D, et al. An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR[J]. J Microbiol Methods, 2006, 65(1):38-48. pmid: 16099520