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On-line Access: 2021-02-07

Received: 2020-08-10

Revision Accepted: 2020-10-08

Crosschecked: 2021-01-06

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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Huixia SHOU

https://orcid.org/0000-0001-6890-5672

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Journal of Zhejiang University SCIENCE B 2021 Vol.22 No.2 P.99-111

http://doi.org/10.1631/jzus.B2000465


Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing


Author(s):  Qi WANG, Xiaoxia SHEN, Tian QIU, Wei WU, Lin LI, Zhi'an WANG, Huixia SHOU

Affiliation(s):  State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China; more

Corresponding email(s):   huixia@zju.edu.cn

Key Words:  DNA extraction, Cellulose filter paper, Field test, Genetically modified (GM) crops, Chinese medicine ultra-fine powder, Rapid molecular identification


Qi WANG, Xiaoxia SHEN, Tian QIU, Wei WU, Lin LI, Zhi'an WANG, Huixia SHOU. Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing[J]. Journal of Zhejiang University Science B, 2021, 22(2): 99-111.

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doi="10.1631/jzus.B2000465"
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%A Qi WANG
%A Xiaoxia SHEN
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%A Wei WU
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Abstract: 
Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification. In this study, a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin (PVC) sheet. This modified method is named EZ-D, for EASY DNA extraction. Compared with the original cetyl trimethylammonium bromide (CTAB) method, DNA extracted by EZ-D is more efficient in polymerase chain reaction (PCR) amplification due to the more stable performance of the EZ-D stick. The EZ-D method is also faster, easier, and cheaper. PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples. A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/μL. Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80% in GC content. EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues. Moreover, when EZ-D was combined with the loop-mediated isothermal amplification (LAMP) method, DNA identification of biological samples could be achieved without the need for specialized equipment. As an optimized DNA purification method, EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.

一种用于实验室和现场检测的高效的植物DNA提取方法的评价及其应用

目的:本研究旨在开发一种优化的滤纸法快速纯化DNA技术--EZ-D(EASY DNA),并将EZ-D与聚合酶链式反应(PCR)/环介导等温扩增(LAMP)技术相结合应用于实验室或室外不同类型植物材料的目的基因的快速鉴定,同时对EZ-D方法的DNA提取效率和所提取DNA的完整性和可扩增能力进行评价。
创新点:EZ-D试纸条采用PVC塑料板支撑纤维素滤纸,使滤纸在DNA提取过程更加稳定和高效。将滤纸快速提取DNA的技术与PCR/LAMP核酸扩增技术相结合,可应用于转基因植物材料外源基因的鉴定和中药超微粉的鉴伪。对滤纸所吸附的DNA浓度进行了评价。基于EZ-D得到的PCR产物的片段长度和GC含量,评价了经滤纸快速提取的DNA的完整度和可扩增能力。
方法:通过比较EZ-D试纸条和文献报道试纸条提取的DNA的扩增结果来评价EZ-D试纸条的优势;通过比较EZ-D和CTAB(十六烷基三甲基溴化铵)方法所提取DNA的扩增结果以及DNA提取时间来评价EZ-D提取DNA的效率;通过单个EZ-D试纸条所吸附的DNA可进行PCR的次数,来评价滤纸吸附的DNA浓度;通过PCR扩增具有不同GC含量或不同长度的基因片段来评价EZ-D提取的DNA的可扩增能力和完整度;使用EZ-D提取不同类型植物材料的DNA,通过观察目的基因的PCR扩增结果来评价EZ-D提取方法的可适用范围。
结论:相较于已报道的滤纸条,EZ-D试纸条对植物DNA的提取更高效和稳定;相较于CTAB法,EZ-D试纸条对植物材料DNA的提取所花时间更短,且DNA的扩增效率相近;一个EZ-D试纸条吸附的DNA可以进行至少16次PCR;EZ-D所提取的DNA可用于扩增片段长度达3000bp或GC含量高达80%的基因片段;EZ-D与PCR相结合可对多种植物材料的目的片段进行快速鉴定。

关键词:DNA提取;纤维素滤纸;转基因作物;田间检测;中药超微粉;快速分子鉴定

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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