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An easy, rapid, and cost-effective method for DNA extraction from various lichen taxa and specimens suitable for analysis of fungal and algal strains.

ABSTRACT: Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples.

SUBMITTER: Park SY

PROVIDER: S-EPMC4298833 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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An easy, rapid, and cost-effective method for DNA extraction from various lichen taxa and specimens suitable for analysis of fungal and algal strains.

Park Sook-Young SY Jang Seol-Hwa SH Oh Soon-Ok SO Kim Jung A JA Hur Jae-Seoun JS

Mycobiology 20141231 4

Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell ...[more]

PMID: 25606001

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Project description:This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcus-like green alga (Chlorophyta) newly isolated from soil collected on Signy Island (maritime Antarctica) in growth medium supplemented with 100 µg/mL cycloheximide (CHX, a widely used antibiotic active against most eukaryotes). In order to test the generality of CHX resistance in taxa originally identified as members of Stichococcus (the detailed taxonomic relationships within this group of algae have been updated since our study took place), six strains were studied: two strains isolated from recent substrate collections from Signy Island (maritime Antarctica) ("Antarctica" 1 and "Antarctica" 2), one isolated from this island about 50 years ago ("Antarctica" 3) and single Arctic ("Arctic"), temperate ("Temperate") and tropical ("Tropical") strains. The sensitivity of each strain towards CHX was compared by determining the minimum inhibitory concentration (MIC), and growth rate and lag time when exposed to different CHX concentrations. All strains except "Temperate" were highly resistant to CHX (MIC > 1000 µg/mL), while "Temperate" was resistant to 62.5 µg/mL (a concentration still considerably greater than any previously reported for algae). All highly resistant strains showed no significant differences in growth rate between control and treatment (1000 µg/mL CHX) conditions. Morphological examination suggested that four strains were consistent with the description of the species Stichococcus bacillaris while the remaining two conformed to S. mirabilis. However, based on sequence analyses and the recently available phylogeny, only one strain, "Temperate", was confirmed to be S. bacillaris, while "Tropical" represents the newly erected genus Tetratostichococcus, "Antarctica 1" Tritostichococcus, and "Antarctica 2", "Antarctica 3" and "Arctic" Deuterostichococcus. Both phylogenetic and CHX sensitivity analyses suggest that CHX resistance is potentially widespread within this group of algae.

Project description:BackgroundRetrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffin-embedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens.Principal findingsFor effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll™ Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and -RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and -RNA than the two other methods, and particularly provides higher quality microRNAs and genomic DNA for subsequent molecular analyses.SignificanceWe determined that co-extraction of genomic DNA and total RNA from a single FFPE specimen is an effective recovery approach to obtain high-quality material for parallel molecular and high-throughput analyses. Our optimized approach provides the option of collecting DNA, which would otherwise be discarded or degraded, for additional or subsequent studies.

Dataset Information

An easy, rapid, and cost-effective method for DNA extraction from various lichen taxa and specimens suitable for analysis of fungal and algal strains.

Publications

An easy, rapid, and cost-effective method for DNA extraction from various lichen taxa and specimens suitable for analysis of fungal and algal strains.

Similar Datasets

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