Abstract
Several commonly used extraction procedures and commercial kits were compared for extraction of DNA from opium poppy (Papaver somniferum L.) seeds, ground seeds, pollen grains, poppy seed filling from a bakery product, and poppy oil. The newly developed extraction protocol was much simpler, reduced the cost and time required for DNA extraction from the native and ground seeds, and pollen grains. The quality of extracted DNA by newly developed protocol was better or comparable to the most efficient ones. After being extended by a simple purification step on a silica membrane column, the newly developed protocol was also very effective in extracting of poppy DNA from poppy seed filling. DNA extracted from this poppy matrix was amplifiable by PCR analysis. DNA extracted from cold-pressed poppy oil and suitable for amplifications was obtained only by methods developed previously for olive oil. Extracted poppy DNA from all tested matrices was analysed by PCR using primers flanking a microsatellite locus (156 bp) and two different fragments of the reference tubulin gene (553 bp and 96 bp). The long fragment of the reference gene was amplified in DNA extracted from native seeds, ground seeds, and pollen grains. Poppy DNA extracted from the filling of bakery product was confirmed only by amplification of short fragments (96 bp and 156 bp). DNA extracted from cold-pressed poppy oil was determined also only by amplification of these two short fragments.
Highlights
Extraction of nucleic acids from various matrices is the first and crucial step in analysis of biological materials generally
Protocols tested for extraction of DNA from native and ground poppy seeds, pollen grains, poppy seed filling from the bakery product, and poppy oil have been differently effective and suitable depending on individual poppy seed matrices or products
DNA from seeds, ground seeds and pollen grains extracted by almost all extraction procedures had quantity and quality sufficient for PCR analysis of short microsatellite marker (156 bp) and long fragment of the reference gene
Summary
Extraction of nucleic acids from various matrices is the first and crucial step in analysis of biological materials generally. Methods of DNA extraction have evolved over time [1], but still contain several basic and necessary steps such as cell disruption, removal of undesirable molecules (lipids, proteins, polyphenols, and others), and purification. Extraction of DNA from young, fast-growing, and healthy tissues is much easier. It is often necessary to extract DNA from plant tissues rich in polysaccharides, lipids, secondary metabolites, or even from very complex matrices (processed seeds, oils, foods, feeds). This is the case of oilseeds where extraction of DNA is considered more demanding than from vegetative plant tissues (e.g., young leaves). Lipids usually prevent the action of solvents during removal of polysaccharides and phenolic
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