Location: Floral and Nursery Plants ResearchTitle: Using acetone for rapid PCR-amplifiable DNA extraction from recalcitrant woody plant taxa
|GUO, YONGHONG(HENRY) - Rutgers University|
Submitted to: Applications in Plant Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/17/2020
Publication Date: 12/3/2020
Publication URL: https://handle.nal.usda.gov/10113/7709375
Citation: Gouker, F.E., Guo, Y., Pooler, M.R. 2020. Using acetone for rapid PCR-amplifiable DNA extraction from recalcitrant woody plant taxa. Applications in Plant Sciences. 8(12):e114-3. https://doi.org/10.1002/aps3.11403.
Interpretive Summary: Quick and effective DNA extraction from plants for genetic analysis can be difficult because some plant taxa contain compounds that can inhibit downstream analyses. ARS scientists in Beltsville, MD developed a simplified DNA extraction protocol using acetone-fixed leaf material that resulted in improved DNA extraction from fresh, frozen, and dried plant leaf samples. This protocol demonstrates the efficacy of tissue preservation and subsequent extraction in acetone with only the addition of 1% SDS without additional alcohol precipitation steps. This method provides a low-cost and rapid alternative to extract DNA with yield and quality that are suitable for downstream PCR applications using fresh, frozen, or oven-dried tissue samples across many herbaceous and woody plant families.
Technical Abstract: Quick and effective DNA extraction from plants for subsequent PCR amplification is sometimes difficult when working across diverse plant taxa that may contain a variety of inhibitory compounds. Multiple and sometimes time-consuming methods may be needed to overcome inhibitory effects and to extract DNA from leaf samples preserved from various field collection methods. The use of acetone in DNA extraction was tested from fresh, frozen, oven-dried, acetone-fixed, and herbarium leaf material of 22 species from 17 woody and herbaceous plant families. A simplified DNA extraction protocol was developed using acetone-fixed leaf material that resulted in improved DNA extraction. The addition of 1% SDS solution resulted in optimal extraction for all tissue samples. Resulting DNA from all extraction protocols was evaluated using both standard PCR and real-time PCR assays. The protocol described here resulted in DNA of sufficient quality and quantity for PCR amplification of recalcitrant plant species as indicated by lowered Ct (threshold cycle) values from real-time assays. This method is simple, fast, and cost-effective and is a reliable tool to extract DNA from recalcitrant woody plant material that contains PCR inhibitors as well as herbaceous plant samples.