Extraction of Plant DNA Made Easy

By Trillium1946 at en.wikipedia (Transferred from en.wikipedia) [Public domain], from Wikimedia Commons

My one attempt at working with plant DNA when I was at the lab bench was trying to create a shotgun library from a rice BAC. Never have I needed to isolate nucleic acid from the source material, but based on my conversations with plant scientists, it can be problematic endeavor between the tough tissue and the compounds that can copurify during extraction and inhibit downstream applications. And if you want to isolate DNA or DNA from plant samples in an automated format, that just adds to the difficulties. Here I review an Applications in Plant Sciences article that compares DNA isolation using the Maxwell® 16 System with two other methods on 25 different plant species samples. The authors note that Promega provided the Maxwell® 16 instrument, DNA isolation cartridges and advice on its use.

University of Wisconsin—Madison researchers started with leaf samples of 25 different species from Wisconsin forests including white trillium (Trillium grandiflorum), twoleaf miterwort (Mitella diphylla) and white snakeroot (Ageratina altissima). Nine millimeter punches were taken from each of four individual samples from each species, three replicates per sample used for each of the three isolation methods: Maxwell® 16 System (paramagnetic cellulose bead), Qiagen DNeasy Plant Mini Kit (silica column) and cetyltrimethylammonium bromide (CTAB)-based extraction. The leaf samples were homogenized using bead beating and suspended in the method’s respective lysis or extraction buffers. Purified DNA samples were either eluted in 100µl of buffer, two passes of elution buffer at 50µl each for the DNeasy and Maxwell® 16 methods, or the pellet suspended in 100µl of TE buffer.

For this DNA isolation method comparison, Moeller et al. were interested in DNA yield, DNA purity and how well the extracted DNA amplified in qPCR. To determine yield, DNA was assessed using gel electrophoresis, optical density measurements on a NanoDrop® spectrophotometer and a fluorescent double-stranded DNA (dsDNA)-binding dye. The Maxwell® 16-extracted samples had an overall yield twice that of DNeasy and CTAB across all 25 species sampled (38ng/µl versus 17 ng/µl). In fact, the Maxwell® 16-based method had higher average yields 17 species, nine of which were noted as significantly higher by researchers. For the other eight species, CTAB extraction showed higher yield. Furthermore, regression analysis of the Maxwell® 16 System and the DNeasy Plant Mini Kit showed the Maxwell® 16 method had a greater absolute yield, and for species that had low absolute yields, a markedly greater relative yield compared to the DNeasy Kit. This reflects the assertion that cellulose-based particles like those in the Maxwell® 16 extraction kit have greater binding capacity compared to silica.

DNA purity was assessed using UV absorbance measurements at A260/A280 with ~1.8 defined as pure DNA. The researchers found that the Maxwell® 16 method had higher purity compared to the DNeasy Kit, but no significant difference between either kit and the CTAB method. In fact, CTAB had the highest A260/A280 ratio of all the methods but eight species had elevated ratios, suggesting contamination affected the absorbance reading. For the A260/A230 ratio, the numbers were similar for all three extraction methods. Moeller et al. found only two plant species DNA extracted by the Maxwell® 16 System showed a ratio of A260/A230 < 1.0 compared to four species for CTAB and seven for the DNeasy Kit. (Note: A ratio < 1.8 indicates organic compound contamination.) In fact, the Maxwell® 16 System showed the greatest consistency in purity ratios among all three extraction methods.

However, the final test of DNA quality is using the purified DNA in PCR assay where contamination can negatively affect the quantity of amplified DNA. Extracted DNA from 12 of the plant species was tested in qPCR, and greatest yield of amplifiable DNA came from the Maxwell® 16 System, significantly more amplified in five species and nine species compared to the DNeasy and CTAB methods, respectively. Maxwell® 16 System amplified twice as much DNA versus DNeasy in eight of the species DNA samples.

After extracting DNA from 25 species of flowering plants found in Wisconsin forests, researchers at the University of Wisconsin—Madison found that the Maxwell® 16 System offered several advantages compared to DNeasy Plant Mini Kit and the CTAB extraction method. The Maxwell® 16 Plant DNA Kit was able to isolate DNA at a higher yield with more amplifiable DNA even in samples with low genomic DNA concentration all in an automated system that can save time and labor. Scientists looking to isolate DNA from plant samples have another option, one that offers consistency and purity for an array of sample species.

Reference
Moeller, J.R., Moehn, N.R., Waller, D.M. and Givnish, T.J. (2014) Paramagnetic Cellulose DNA Isolation Improves DNA Yield and Quality Among Diverse Plant Taxa, Applications in Plant Sciences, 2 1400048. DOI: http://dx.doi.org/10.3732/apps.1400048

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Sara Klink

Technical Writer at Promega Corporation
Sara is a native Wisconsinite who grew up on a fifth-generation dairy farm and decided she wanted to be a scientist at age 12. She was educated at the University of Wisconsin—Parkside, where she earned a B.S. in Biology and a Master’s degree in Molecular Biology before earning her second Master’s degree in Oncology at the University of Wisconsin—Madison. She has worked for Promega Corporation for more than 15 years, first as a Technical Services Scientist, currently as a Technical Writer. Sara enjoys talking about her flock of entertaining chickens and tries not to be too ambitious when planning her spring garden.

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