Isolation of High-Quality DNA from a Desert Plant Reaumuria soongorica

Wang, Xiaohua; Xiao, Hansong; Zhao, Xinquan; Li, Caizhi; Ren, Juan; Wang, Fang; Pang, Lei

doi:10.5772/38367

Cited by 4 publications

(7 citation statements)

References 27 publications

(27 reference statements)

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“…Commercial DNA isolation kits (Table 2) have proven effective in isolating genomic DNA from leaves of many crop plants, such as rice, barley, tomato, citrus, and Arabidopsis [30-32]. However, these kits were not effective to isolate useful amounts of high quality genomic DNA from dry papaya seeds (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

“…However, these kits were not effective to isolate useful amounts of high quality genomic DNA from dry papaya seeds (Figure 1A). Genomic DNA isolation from plants is often recalcitrant [32-34], and difficulties in obtaining high quality genomic DNA from papaya tissues (e.g., leaf, fruit) have also been encountered by other researchers [24,35-37]. Proteins, polysaccharides, polyphenols, fibers, carbohydrates, lipids and other various secondary metabolites can decrease the efficiency of plant DNA extraction [38-40].…”

Section: Resultsmentioning

confidence: 99%

“…Some, if not most, of these constituents are likely causes of DNA extraction problems in papaya seed [41]. Endonucleases, polyphenols, or polysaccharides, which can all co-precipitate along with DNA during extraction, result in irreversible interactions with nucleic acids that can further affect purification of DNA [34,38] and enzymatic reactions such as Taq DNA polymerase-mediated PCR [32,42,43]. Thus, it is not unusual for DNA extraction methods to require species- and tissue-specific optimization [34,44,45].…”

Section: Resultsmentioning

confidence: 99%

“…It is generally regarded that ratio A260/A280 values of 1.8 indicate high purity DNA, whereas less than 1.8 indicate protein contamination in DNA samples, and more than 1.8 indicate that there might be RNA contamination [32]. The resultant A260/A280 and A260/A230 absorbance ratios were 1.82 and 1.76 respectively in the modified CTAB method, indicating that the papaya seed genomic DNA was free of protein and polysaccharides/polyphenol contamination (Table 2) and the quantity and quality of genomic DNA was suitable for both conventional as well as real-time PCR amplifications.…”

Section: Resultsmentioning

confidence: 99%

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Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papayaL.)

et al. 2013

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BackgroundGenetically engineered (GE) ringspot virus-resistant papaya cultivars ‘Rainbow’ and ‘SunUp’ have been grown in Hawai’i for over 10 years. In Hawai’i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities.ResultsWe assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/μl of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%.ConclusionsThis method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai’i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papayaL.)

et al. 2013

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“…A single DNA isolation protocol is not likely to be applicable for all the plant tissues (Loomis 1974). Most of the cetyl trimethylammonium bromide (CTAB)-based protocols used for the extraction of DNA were tailored according to the internal components of each single plant species (Wang et al 2012;Moreira and Oliveira 2011).…”

Section: Introductionmentioning

confidence: 99%

Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method

2019

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Background: Reliable measurement of DNA concentration and purity is important for almost all molecular genetics studies. Different plant species have varying levels of polysaccharides, polyphenols, and other secondary metabolites which combine with nucleic acids during DNA isolation and further affect the quality of the extracted DNA. The current extraction protocol is based upon the conventional cetyl trimethylammonium bromide (CTAB) method with further modifications for the extraction of DNA from variable plant seeds and crops belonging to seven different orders. The principle modifications currently employed for DNA extraction involved the use of higher CTAB concentration and higher levels of 2-β-mercaptoethanol. Additionally, higher concentrations of sodium chloride and potassium acetate were added simultaneously with absolute ice cold isopropanol for the precipitation of DNA free from polysaccharides. Results and conclusion: The prescribed modifications in the present method establish a quick and efficient standardized protocol for DNA extraction from different plant orders. The current extraction protocol, therefore, can be of great value for molecular analysis involving large numbers of different plant samples from different orders. These modifications consistently produced pure and high-quality DNA suitable for further molecular analysis. Successful PCR amplification with random amplified polymorphic DNA primer, NPTII gene, and the complete digestion of the isolated DNA with the HindIII restriction enzyme validated the quality of the isolated DNA. Moreover, it reflects the efficiency of the protocol and proves its suitability for further applications for the assessment of food safety, detection of genetically modified (GM) crops, and conservation of biodiversity.

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