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Abstract PremiseThe preservation of plant tissues in ethanol is conventionally viewed as problematic. Here, we show that leaf preservation in ethanol combined with proteinase digestion can provide high‐quality DNA extracts. Additionally, as a pretreatment, ethanol can facilitate DNA extraction for recalcitrant samples. MethodsDNA was isolated from leaves preserved with 96% ethanol or from silica‐desiccated leaf samples and herbarium fragments that were pretreated with ethanol. DNA was extracted from herbarium tissues using a special ethanol pretreatment protocol, and these extracts were compared with those obtained using the standard cetyltrimethylammonium bromide (CTAB) method. ResultsDNA extracted from tissue preserved in, or pretreated with, ethanol was less fragmented than DNA from tissues without pretreatment. Adding proteinase digestion to the lysis step increased the amount of DNA obtained from the ethanol‐pretreated tissues. The combination of the ethanol pretreatment with liquid nitrogen freezing and a sorbitol wash prior to cell lysis greatly improved the quality and yield of DNA from the herbarium tissue samples. DiscussionThis study critically reevaluates the consequences of ethanol for plant tissue preservation and expands the utility of pretreatment methods for molecular and phylogenomic studies.
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Optimizing the lysis step in CTAB DNA extractions of silica‐dried and herbarium leaf tissues
Abstract Premise The use of cetyltrimethylammonium bromide (CTAB) is an effective and inexpensive method of extracting DNA from plants. The CTAB protocol is frequently modified to optimize DNA extractions, but experimental approaches rarely perturb a single variable at a time to systematically infer their effect on DNA quantity and quality. Methods and Results We investigated how chemical additives, incubation temperature, and lysis duration affected DNA quantity and quality. Altering those parameters influenced DNA concentrations and fragment lengths, but only extractant purity was significantly affected. CTAB and CTAB plus polyvinylpyrrolidone buffers produced the highest DNA quality and quantity. Extractions from silica gel–preserved tissues had significantly higher DNA yield, longer DNA fragments, and purer extractants compared to herbarium‐preserved tissues. Conclusions We recommend DNA extractions of silica gel–preserved tissues that include a shorter and cooler lysis step, which results in purer extractions compared to a longer and hotter lysis step, while preventing fragmentation and reducing time.
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- Award ID(s):
- 2117446
- PAR ID:
- 10451107
- Date Published:
- Journal Name:
- Applications in Plant Sciences
- Volume:
- 11
- Issue:
- 3
- ISSN:
- 2168-0450
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Cetyltrimethylammonium bromide (CTAB)–based methods are widely used to isolate DNA from plant tissues, but the unique chemical composition of secondary metabolites among plant species has necessitated optimization. Research articles often cite a “modified” CTAB protocol without explicitly stating how the protocol had been altered, creating non‐reproducible studies. Furthermore, the various modifications that have been applied to the CTAB protocol have not been rigorously reviewed and doing so could reveal optimization strategies across study systems. We surveyed the literature for modified CTAB protocols used for the isolation of plant DNA. We found that every stage of the CTAB protocol has been modified, and we summarized those modifications to provide recommendations for extraction optimization. Future genomic studies will rely on optimized CTAB protocols. Our review of the modifications that have been used, as well as the protocols we provide here, could better standardize DNA extractions, allowing for repeatable and transparent studies.more » « less
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Abstract Next‐generation sequencing has greatly expanded the utility and value of museum collections by revealing specimens as genomic resources. As the field of museum genomics grows, so does the need for extraction methods that maximize DNA yields. For avian museum specimens, the established method of extracting DNA from toe pads works well for most specimens. However, for some specimens, especially those of birds that are very small or very large, toe pads can be a poor source of DNA. In this study, we apply two DNA extraction methods (phenol–chloroform and silica column) to three different sources of DNA (toe pad, skin punch and bone) from 10 historical avian museum specimens. We show that a modified phenol–chloroform protocol yielded significantly more DNA than a silica column protocol (e.g., Qiagen DNeasy Blood & Tissue Kit) across all tissue types. However, extractions using the silica column protocol contained longer fragments on average than those using the phenol–chloroform protocol, probably as a result of loss of small fragments through the silica column. While toe pads yielded more DNA than skin punches and bone fragments, skin punches proved to be a reliable alternative source of DNA and might be especially appealing when toe pad extractions are impractical. Overall, we found that historical bird museum specimens contain substantial amounts of DNA for genomic studies under most extraction scenarios, but that a phenol–chloroform protocol consistently provides the high quantities of DNA required for most current genomic protocols.more » « less
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Abstract BackgroundThere is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications. ResultsIn a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant,Arabidopsis thaliana(L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastidrbcLand nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis. ConclusionsThe method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1002/aps3.11522
