Fig. 1 EZ-D stick is more efficient for PCR amplification than the dipstick reported by
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State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou310058, China
1.State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou310058, China
2.Zhejiang Institute of Chinese Medicine, Hangzhou310023, China
3.Shanghai YouLong Biotech Co., Ltd., Shanghai200063, China
Published: 15 February 2021 ,
Received: 10 August 2020 ,
Revised: 08 October 2020 ,
Accepted: 06 January 2021
Cite this article
WANG Qi,SHEN Xiaoxia,QIU Tian et al., 2021. Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(2):99-111.
WANG Qi,SHEN Xiaoxia,QIU Tian et al., 2021. Evaluation and application of an efficient plant DNA extraction protocol for laboratory and field testing. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(2):99-111. DOI: 10.1631/jzus.B2000465.
State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou310058, China
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.
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。
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。
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。
Nucleic acid-based molecular diagnosis assays have increased in use in many fields in recent years, due to their outstanding sensitivity, specificity, and speed (
Current rapid extraction methods include alkaline lysis and direct-polymerase chain reaction (PCR), both of which shorten the DNA extraction to about 10 min. However, these methods still require controlled heating, centrifugation, and other operations (
Here, we present a modified Z-Dipstick plant DNA extraction method named EASY DNA (EZ-D). Instead of imbedding the handle of the test strip with paraplast, a polyvinyl chloride (PVC) resin material is used as the handle of the dipstick, as well as the support of the DNA-binding zone. EZ-D has been successfully used for DNA extraction from a variety of plant species and plant tissues and the DNA is suitable for a wide range of downstream applications including PCR and loop-mediated isothermal amplification (LAMP). The method has great prospects for application in the field or market for easy and fast molecular identification.
Six different types of transgenic plants and their corresponding non-transgenic controls were used in this study (
Plant species | Name of the material | Type of the material | Content of the transgene cassette |
---|---|---|---|
Oryza sativa | T51 | Transgenic rice | 35S::cry1Ab/Ac; 35S::HPT |
MH63 | Non-transgenic control | None | |
Zea mays | ZC1 | Transgenic maize | 35S::cry1Ab; 35S::g10-epsps |
HiⅡ | Non-transgenic control | None | |
Glycine max | GC1 | Transgenic soybean | 35S::cry1Ab; 35S::cp4-epsps |
Wandou28 | Non-transgenic control | None | |
Glycine max | SWEET15 | Transgenic soybean | 35S::bar |
Williams82 | Non-transgenic control | None | |
Glycine max | ZUTS-33 | Transgenic soybean | 35S::g10-epsps |
HC-3 | Non-transgenic control | None | |
Fritillaria unibracteata | CB1 | Chinese medicine | Without ZB1 gene |
Fritillaria przewalskii | CB2 | Chinese medicine | Without ZB1 gene |
Fritillaria delavayi | CB3 | Chinese medicine | Without ZB1 gene |
Fritillaria thunbergii | ZB | Chinese medicine | With ZB1 gene |
Whatman cellulose filter paper No. 1 (Whatman, UK) was cut into strips measuring 4 mm×20 cm. A commercial self-adhesive PVC sheet (YouLong Biotech, China) was cut into strips of 44 mm×20 cm. Four millimeters of tape on the bottom of the plastic sheet was peeled off manually and then a filter paper strip was attached to the exposed adhesive area. The plastic sheet with attached filter paper was cut into strips of 2 mm width to form the final EZ-D sticks (
Fig. 1 EZ-D stick is more efficient for PCR amplification than the dipstick reported by
To avoid contamination, filter papers were sterilized prior to the preparation. Only the PVC handle side of EZ-D stick was touched during the preparation. The ready EZ-D sticks were further treated with ultraviolet (UV)-light for 30 min, and then packed.
The protocol for rapid DNA extraction from biological samples using EZ-D is presented in
A TL2010S high throughput ball mill (DHS Life Science & Technology, China) was used for grinding a large number of samples or hard samples, such as the needle leaves of larch. In addition, we also tested drilling soybean seeds with a miniature electric drill fitted with a 1-mm diameter bit (P-500-3, Slite, USA) to obtain seed powder from the cotyledons, which allowed the seeds to retain their viability (
After disruption, the EZ-D stick was dipped into the plant tissue lysate for 10 s and then transferred into 200 μL of wash buffer (10 mmol/L Tris (pH 8.0), 0.1% Tween-20) for 5 s. The stick was then dipped into the DNA amplification reaction system for 5 s, to elute the DNA. The whole protocol took about 30 s, after which the purified DNA was ready for amplification or other molecular identification.
The quality of the DNA extracted by EZ-D was evaluated by a NanoDrop 1000 Lite spectrophotometer (Thermo Scientific, USA). The DNA absorbed by the EZ-D stick was finally released by dipping the stick into 20 μL of double-distilled water (ddH2O) for 5 s. In this way, we could evaluate the quality ofDNA released into the PCR solution. The UV absorption peaks of DNA, proteins, and polysaccharides were at 260, 280, and 230 nm, respectively. The ratios of absorbance at 260 nm to 280 nm (A260/A280) and A260/A230 were recorded to evaluate the purity of DNA against proteins and polysaccharides, respectively (
A modified CTAB method was used as the control, to represent a conventional DNA extraction method. One hundred milligrams of plant samples were finely ground using liquid nitrogen. The plant powder was mixed with 700 μL of extraction buffer (2% (20 g/L) CTAB, 0.7 mol/L NaCl, 10 mmol/L EDTA, 50 mmol/L Tris-HCl (pH 8.0), 0.5% β-mercaptoethanol, which was preheated at 65 ℃). After 1.5 h at 65 ℃, 700 μL of chloroform was added to the mixture which was rotated gently at room temperature, followed by centrifugation at 12 000 r/min for 10 min. Three hundred microliters of supernatant was transferred to a new tube and mixed gently with 210 μL of isopropanol. After the DNA precipitated, the flocculent DNA was removed and transferred to a new tube and washed twice with 1 mL 75% ethanol. After washing, the DNA was suspended in 100 μL of ddH2O and quantified using the NanoDrop 1000 Lite spectrophotometer (Thermo Scientific).
Genomic DNA samples purified by the CTAB method were diluted in series by a factor of 10, with final concentrations ranging from 100 to 0.01 ng/μL. For the detection of PCR sensitivity of EZ-D DNA, dilutions of DNA were prepared by adding 1 μL of genomic DNA with diluted concentrations onto the DNA-binding zone of the EZ-D sticks, followed by washing. The resulting sticks were immersed into a PCR reaction mixture for testing.
Specific primers for each of the genes were designed using the Oligo software (Version 7.0, Molecular Biology Insights, USA). The genes, primer sequences, corresponding amplicon lengths, and GC contents of the amplicons used for the tests are listed in
Gene | Primer name | Primer sequence (5'→3') | GC content (%) | Amplicon length (bp) |
---|---|---|---|---|
bar | bar-F | GTCAACCACTACATCGAGACAAGC | 68 | 260 |
bar-R | AGCAGGTGGGTGTAGAGCGT | |||
cry1Ab/Ac | cry1Ab/Ac-F | GGCCATACAACTGCTTGAGT | 49 | 1000 |
cry1Ab/Ac-R | GCGTTTCCCATAGTTCCATA | |||
cry1Ab | cry1Ab-F | GACAACAACCCCAACATCAACGAGTG | 65 | 583 |
cry1Ab-R | GGTCGTTGTAGCGGCTGTTGATGGT | |||
cp4-epsps | cp4-epsps-F | TGATCCTCACGCTTCAGGAGATGGGAGC | 63 | 488 |
cp4-epsps-R | CAGATCCATGAACTCTGGGAAGCTCGTC | |||
g10-1* | g10-1-F | TCTAAGGTCATCGGAGGCAGTAGCT | 54 | 604 |
g10-1-R | TAGAGCACAGCCATCCAAGAACTAC | |||
g10-2* | g10-2-F | TCTCACCTCTGGCACCACTTTCG | 70 | 703 |
g10-2-R | CCACGTTCTCCCAGGTGGTGT | |||
g10-3* | g10-3-F | TTTGATGAGCCTTTGTACACAGC | 39 | 468 |
g10-3-R | AAGCAATAAAATAAAATGAAGACAAGGT | |||
g10-epsps | 33-F | TAATTCGGGGGATCTGGATTTTAGTA | ||
33-R1 | CTCTTAGACTCAAGGAATGCGATAGA | 44 | 500 | |
33-R2 | AGTACGCTTCCGCCCTTATGTTCCTC | 48 | 1000 | |
33-R3 | CTCTTCCAGCTACCTTCGACGTTATC | 50 | 1500 | |
33-R4 | GATGTGATATCTCCACTGACGTAAGG | 48 | 2000 | |
33-R5 | TCAAAAGGACAGTAGAAAAGGAAGGT | 48 | 2500 | |
33-R6 | GGGTTTTCCCAGTCACGACGTTGTAA | 48 | 3000 | |
GmActin | Actin-F | TTTGATGAGCCTTTGTACACAGC | ||
Actin-R1 | AAGCAATAAAATAAAATGAAGACAAGGT | 41 | 500 | |
Actin-R2 | ACCATCCTTAACATTATTTATGT | 37 | 1000 | |
Actin-R3 | AGCATCGTCACCAGCAAATCCTG | 37 | 1500 | |
Actin-R4 | ATAGATTGGTACAGTGTGACTCA | 39 | 2000 | |
Actin-R5 | GGTGGAGCTACAACCTTAATCTT | 40 | 2500 | |
Actin-R6 | TACAAAAAGAAAATGGAGGAAAAAGAGAT | 40 | 3000 | |
Actin-F7 | TCAATGTGCCTGCCATGTATGTG | 44 | 246 | |
Actin-R7 | CCAGCGAGATCCAAACGAAG | |||
HPT | HPT-F | GAAGTGCTTGACATTGGGGAGT | 58 | 472 |
HPT-R | AGATGTTGGCGACCTCGTATT | |||
OsbHLH156 | 156-F | GGTCGCCGCGCTCGAGGAGGC | 80 | 360 |
156-R | GCCACGACGCGGTCCGGCGAG | |||
trnL-trnF | trnL-trnF-F | GGGGATAGAGGGACTTGAAC | 38 | 540 |
trnL-trnF-R | CGAAATCGGTAGACGCTACG | |||
ZB1 | ZB1-F | GCCCTTGAAAGATTCTGGTA | 43 | 207 |
ZB1-R | AGTTGGTGGACTCACTGG | |||
35S | 35S-F | GCTCCTACA AATGCCATCA | 50 | 195 |
35S-R | GATAGTGGG ATTGTGCGTCA |
*g10-1, g10-2, and g10-3 are three different fragments of g10-epsps.
PCR reactions were performed in a total volume of 20 μL in a reaction mix that contained 10 μL of 2× Taq Master Mix Kit (CWBiotech, China) or PrimeSTAR® Premix (TaKaRa Biotech, Japan), 0.4 μL of 10 μmol/L primers, 8.2 μL of ddH2O, and 1 μL of template DNA (the volume of template DNA transferred from the EZ-D stick was defaulted at 1 μL). The PCR cycling parameters were as follows: initial denaturation at 95 ℃ for 2 min, and then 40 cycles of denaturation at 95 ℃ for 20 s, 55‒60 ℃ for 20 s, 72 ℃ for 20 s, followed by final extension of 72 ℃ for 5 min or 98 ℃ for 2 min, 40 cycles of 98 ℃ for 20 s, 55‒60 ℃ for 20 s, 72 ℃ for 1‒3 min, followed by final extension of 72 ℃ for 5 min.
For DNA fragments rich in GC content, the reaction mix consisted of 10 μL 2× GC Buffer Ⅱ, 3.2 μL 2.5 mmol/L deoxyribonucleoside triphosphates (dNTPs), 0.2 μL La Taq (TaKaRa Biotech), 0.2 μL of 10 μmol/L primers, 6.2 μL of ddH2O, and 1 μL of template DNA (the volume of template DNA transferred from the EZ-D stick was defaulted at 1 μL). The PCR cycling parameters were initial denaturation at 94 ℃ for 5 min, followed by 40 cycles of denaturation at 94 ℃ for 30 s, 60 ℃ for 30 s, 72 ℃ for 20 s, and a final extension at 72 ℃ for 2 min.
All the resulting DNA products were separated on a 1% (0.01 g/mL) agarose gel at 150 V for 30 min.
Relevant LAMP primers (
Gene | Primer type* | Gene location (nt) | Primer sequence (5'→3') | Sample for detection |
---|---|---|---|---|
g10-epsps | F3 | 718‒735 | AAGAGGAGCGTGGGACTT | Transgenic soybean ZUTS-33 |
B3 | 926‒944 | GACCTCAGGGTGACCTTCT | ||
FIP | 742‒761/TTTT/784‒805 | CGCTTCCGCCCTTATGTTCCTCTTTTACCGGTGAGTCTAAGTTCGA | ||
BIP | 853‒874/TTTT/896‒915 | TGGACCGGAAACGGAGATAGGGTTTTGAAAGACTTGGTGCTTGGGT | ||
LF | 763‒863 | GGTCCTCTTCTTCCTGACGGA | ||
ZB1 | F3 | 446‒465 | GCCCTTGAAAGATTCTGGTA | Chinese medicine ZB1 |
B3 | 635‒652 | AGTTGGTGGACTCACTGG | ||
FIP | 466‒485/T/507‒526 | ATTGCTACACGGTGATGCCCTATCAACCGTTGAAAAGTTGC | ||
BIP | 533‒556/T/596‒614 | CTAAGGGAGTAGTGAAACTGGTGTTTTTTGGATGCCTGTTCAG |
* F3, B3, FIP, BIP, and LF were forward outer primer (F3) and backward outer primer (B3), forward inner primer (FIP), backward inner primer (BIP), and left loop primer (LF), respectively. LAMP: loop-mediated isothermal amplification.
HNB in the LAMP amplification mixture is as a colorimetric indicator. A color change from violet to sky blue indicates that the tested sample contains the gene of interest (
Equipment-free nucleic acid extraction dipstick methodology using untreated cellulose-based paper to capture nucleic acids was reported by
To demonstrate the high efficiency of the EZ-D stick in rapid DNA extraction, we compared the PCR amplification efficiency of DNA extracted from the EZ-D stick and from the Z-Dipstick. The DNA of 20-mg leaves of ZUTS-33 was extracted using each of these two sticks. The concentration and quality of DNA extracted by the two types of sticks were the same. However, the results showed that significantly more PCR product was obtained by the DNA extracted from the EZ-D stick than from the Z-Dipstick (Figs.
Type | DNA concentration (ng/μL) | A260/A280 | A260/A230 |
---|---|---|---|
Root | 20.59±4.13 | 1.73±0.27 | 0.77±0.01 |
Stem | 21.85±1.68 | 1.50±0.08 | 0.36±0.01 |
Leaf | 24.10±6.05 | 1.41±0.16 | 0.43±0.08 |
Flower | 22.13±5.16 | 1.47±0.15 | 0.58±0.06 |
Seed powder | 9.38±1.86 | 1.50±0.31 | 0.40±0.09 |
Ultrafine powder | 5.82±1.38 | 1.41±0.58 | 0.30±0.05 |
Data are expressed as mean±standard deviation (n=5).
The advantages of the EZ-D method over a conventional CTAB method are listed in
Method | Total time cost | Personal skill | Organic reagent | Electrical device | Cost per sample (CNY) |
---|---|---|---|---|---|
EZ-D | 30-60 s | Low | No | No | 0.2 |
CTAB | 1.5-3.0 h | High | Yes | Yes | 0.9 |
EZ-D: EASY DNA; CTAB: cetyl trimethylammonium bromide.
To assess the quality of DNA extracted by EZ-D, the DNA samples were used to amplify eight DNA fragments of six different genes by PCR. DNA was extracted from leaves of transgenic plants or the corresponding non-transgenic control plants using the EZ-D method as described in
Fig. 2 Molecular identification of DNA components using EZ-D DNA. PCR efficiency (a) and sensitivity (b) were tested using DNA extracted by the CTAB and the EZ-D methods. Plant samples were from several independent transgenic plants including rice line T51, maize line ZC1, soybean lines GC1, SWEET15, and ZUTS-33 with their corresponding non-transgenic controls of MH63 rice, HiII maize, and soybean lines Wandou 28, Williams 82, and HC-3; g10-1 and g10-2 are two fragments of the g10-epsps gene. Details of transgenic genes are listed in the Table 1. The labels of 100, 10, 1, 0.1, and 0.01 at the top of the electropherogram of (b) are the DNA concentrations (ng/μL). (c) DNA extracted by EZ-D was amplified for DNA fragments ranging from 500 to 3000 bp in length. 1‒6, PCR products for amplification of DNA fragments of 500, 1000, 1500, 2000, 2500, and 3000 bp, respectively. M: DL2000 marker; B: blank control; C and E: DNA extracted by CTAB and EZ-D, respectively; N: non-transgenic control. CTAB: cetyl trimethylammonium bromide; EZ-D: EASY DNA.
To determine whether the DNA captured by the EZ-D sticks could be used for multiple PCR assays, a single EZ-D stick was placed in the lysate of the transgenic soybean ZUTS-33 for 10 s, immersed in the wash buffer for 5 s, and finally dipped sequentially into each of 16 PCR tubes containing only water, for 5 s each tube. The 16 tubes were divided into two groups which were used to amplify the transgene g10-epsps and the housekeeping gene GmActin, respectively, by adding the rest of the corresponding components of the PCR reactions. The results showed that the DNA content in a single EZ-D stick could be used for at least 16 PCR reactions (Fig. S2).
To further examine the amplifiability of the EZ-D-extracted DNA and take the GC content of the amplified fragment into consideration, a DNA fragment of OsbHLH156 (
To evaluate the sensitivity of EZ-D sticks, genomic DNA samples extracted by the CTAB method from four different transgenic plants were to be tested. One microgram of the diluted DNA, with final concentrations ranging from 100 to 0.01 ng/μL, was added into the EZ-D sticks and then applied to the PCR system. The first four concentrations of DNA could be amplified, while the 0.01 ng/μL DNA samples from either protocol could not be amplified. Therefore, the PCR detection limit for the DNA samples prepared by the EZ-D method is considered the same as that of samples prepared by the CTAB method, i.e., 0.1 ng/μL(
To evaluate the integrity of DNA extracted by EZ-D, the DNA extracted from the transgenic soybean plant ZUTS-33 using EZ-D was amplified by PCR using primer pairs targeting the soybean GmActin and g10-epsps genes (
To determine if EZ-D is suitable for DNA extraction from various herbaceous plant tissues, samples of roots, stems, leaves, flowers at flowering stage, and dry seeds were collected from transgenic soybean line ZUTS-33 and subjected to the EZ-D and CTAB DNA extraction protocols. The fragments of transgene g10-epsps and the housekeeping gene GmActin were amplified. Results showed that both fragments were successfully amplified from the EZ-D DNA from all five soybean tissues with amplification efficiency similar to those from using CTAB DNA (
Fig. 3 Application of EASY DNA (EZ-D) to a variety of plant samples. (a) Electropherogram of PCR using DNA extracted from different soybean tissues by EZ-D or cetyl trimethylammonium bromide (CTAB) methods. g10-epsps and GmActin are two genes used for the assay; g10-3 is fragment 3 of g10-epsps (Table 2). M, DL1000 marker; N, non-transgenic control; B, blank control; C, DNA extracted by CTAB; E, DNA extracted by EZ-D with three replicates. (b) Electropherogram of PCR using DNA extracted from larch needles by EZ-D. P, positive control; Samples 1‒11, 11 larch leaf samples. (c) Application of EZ-D for molecular identification of ultrafine powdered Chinese medicine. ZB, Zhe Beimu, Fritillaria thunbergii; CB1, Chuan Beimu 1, Fritillaria unibracteata; CB2, Chuan Beimu 2, Fritillaria przewalskii; CB3, Chuan Beimu 3, Fritillaria delavayi; T1‒T5, ultrafine powders of CB containing 5%, 10%, 15%, 20%, and 25% of ZB, respectively; ZB1, a molecular marker of ZB.
Many Chinese medicines produced from plant materials are prepared as an ultrafine powder (
LAMP is one of the most powerful isothermal amplification techniques. It uses four to six primers and Bst DNA polymerase. The LAMP method does not require thermocycling since the amplification is performed at a constant temperature between 60 and 65 ℃ (
Fig. 4 Combination of the EZ-D and LAMP methods for DNA detection of transgenic soybean (a) and ultrafine powder samples of Chinese medicine (b) sampled in field conditions. P: positive control; B: blank control; N: non-transgenic control; Root, stem, leaf, flower, and seed samples were taken from transgenic soybean line ZUTS-33; ZB: Zhe Beimu, Fritillaria thunbergii; CB1: Chuan Beimu 1, Fritillaria. unibracteata; CB2: Chuan Beimu 2, Fritillaria przewalskii; CB3: Chuan Beimu 3, Fritillaria delavayi; T1‒T5, ultrafine powders of CB containing 5%, 10%, 15%, 20%, and 25% of ZB, respectively.
We also tested whether the LAMP-HNB technique can be used for identification of Chinese medicines as described in last section. While no visible color change was observed in the negative control or pure CB samples, CB powders mixed with as little as 5% ZB turned sky blue in the test tube and could be identified (
In this study, we optimized the cellulose filter paper DNA extraction method based on the protocol of
Although the concentration of DNA extracted with EZ-D was relatively low, it could still be sensitively detected by PCR for at least 16 times (Fig. S2). The standard values of A260/A280 and A260/A230 of pure DNA are expected to 1.8‒2.0 and >1.8, respectively (
In recent years, there have been a few reports on the method of rapid DNA purification using cellulose filter paper (
The safety assessment of genetically modified (GM) plants in the field and products in the food market is one of the most important applications of molecular identification (
In this study, we have developed a modified filter paper stick, the EZ-D stick, for rapid DNA extraction from transgenic plants and Chinese medicine ultrafine powder, based on a published method. The EZ-D stick overcomes the three main disadvantages of the published Z-Dipstick, including the complicated production process, ease of contamination, and instability resulting from the loss of PCR compounds from deformed cellulose filter paper. The EZ-D method can extract DNA from a variety of plants and their tissues. The sensitivity of PCR based on the EZ-D extraction method can reach 0.1 ng/μL. Fragments of up to 3000 bp and with a GC content of up to 80% can be amplified. In terms of practicability, EZ-D has realized the application and popularization of cellulose filter paper for rapid DNA purification in laboratories and markets.
Qi WANG and Huixia SHOU designed the study and wrote the manuscript. Qi WANG, Xiaoxia SHEN, Tian QIU, Wei WU, Lin LI, and Zhi'an WANG performed the experiments and interpreted the results.All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
The research was supported by the National Transgenic Major Program of China (No. 2019ZX08010-002), the China Agriculture Research System (No. CARS-21), and the Major Science and Technology Projects of Breeding New Varieties of Agriculture in Zhejiang Province, China (No. 2016C02058).We show our appreciation to Professors Zhicheng SHEN, Jumin TU, and Min WANG of Zhejiang University (Hangzhou, China) for providing plant materials. We also thank Ms. Anita K. SNYDER of Plant Science Copy Editor (USA) for language editing.
Qi WANG, Xiaoxia SHEN, Tian QIU, Wei WU, Lin LI, Zhi’an WANG, and Huixia SHOU declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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