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Comparison of DNA extraction methods for non-marine molluscs: is modified CTAB DNA extraction method more efficient than DNA extraction kits?

ABSTRACT: Isolation of high molecular weight DNA from gastropod molluscs and its subsequent PCR amplification is considered difficult due to excessive mucopolysaccharides secretion which co-precipitate with DNA and obstruct successful amplification. In an attempt to address this issue, we describe a modified CTAB DNA extraction method that proved to work significantly better with a number of freshwater and terrestrial gastropod taxa. We compared the performance of this method with Qiagen^® DNeasy Blood and Tissue Kit. Reproducibility of amplification was verified using a set of taxon-specific primers, wherein modified CTAB extracted DNA could be replicated at least four out of five times but kit extracted DNA could not be replicated. In addition, sequence quality was significantly better with CTAB extracted DNA. This could be attributed to the removal of polyphenolic compounds by polyvinyl pyrrolidone which is the only difference between conventional and modified CTAB DNA extraction methods for animals. The genomic DNA isolated using modified CTAB protocol was of high quality (A260/280 ≥ 1.80) and could be used for downstream reactions even after long-term storage (more than 2 years).

SUBMITTER: Chakraborty S

PROVIDER: S-EPMC6982617 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Comparison of DNA extraction methods for non-marine molluscs: is modified CTAB DNA extraction method more efficient than DNA extraction kits?

Chakraborty Sudeshna S Saha Anwesha A Neelavar Ananthram Aravind A

3 Biotech 20200125 2

Isolation of high molecular weight DNA from gastropod molluscs and its subsequent PCR amplification is considered difficult due to excessive mucopolysaccharides secretion which co-precipitate with DNA and obstruct successful amplification. In an attempt to address this issue, we describe a modified CTAB DNA extraction method that proved to work significantly better with a number of freshwater and terrestrial gastropod taxa. We compared the performance of this method with Qiagen<sup>®</sup> DNeas ...[more]

PMID: 32030338

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Project description:BackgroundDNA isolation and PCR amplification from molluscan taxa is considered as problematic because polysaccharides in tissue and mucus presumably co-precipitate with the DNA and inhibit the activity of DNA polymerase. In the present study we tested two common extraction methods on specimens from the mollusc collection of the Natural History Museum Vienna (NHMW). We analysed a broad variety of taxa covering a large temporal span (acquisition years 1877 to 1999), which distinguishes our study from previous ones where mostly fresh material was used. We also took other factors into account: effects of sample age, effects of formaldehyde treatment and taxon-specific problems. We used several primer combinations to amplify amplicons of different lengths of two mitochondrial genes: cytochrome c oxidase subunit 1 (COI) and 16S rRNA gene (16S).ResultsOverall PCR success was 43 % in the 576 extractions (including all primer combinations). The smallest amplicon (~240 bp) showed the best results (49 % positive reactions), followed by the 400 bp amplicon (40.5 %). Both short sections yielded significantly better results than the 700 bp long amplicon (27 %). Comparatively, the Gen-ial-First, All-tissue DNA-Kit-extraction method performed significantly better than Promega-Tissue and Hair Extraction Kit. Generally, PCR success is age-dependent. Nonetheless, we were able to obtain the longest amplicon even from 137-year-old material. Importantly, formaldehyde traces did not totally inhibit amplification success, although very high concentrations did.ConclusionsMuseum material has gained importance for DNA analysis in recent years, especially for DNA barcoding projects. In some cases, however, the amplification of the standard barcoding region (partial sequence of the COI) is problematic with old material. Our study clearly shows that the COI barcoding region could be amplified in up to 49 % of PCRs (varying with amplicon length), which is, for museum samples, quite a high percentage. The difference between extraction methods was minimal and we recommend using an established kit for a first attempt because experience and routine in handling might be more important than slight performance differences of the various kits. Finally, we identify fixation, storage, sample conservation and documentation of the specimens' history rather than the DNA extraction method to be the most crucial factors for PCR success.

Project description:BackgroundMolluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area.Methodology/principal findingsThis study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0-26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%-46.5%), and showed a significant positive correlation with nearest neighbour distances.Conclusions/significanceDNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.

Dataset Information

Comparison of DNA extraction methods for non-marine molluscs: is modified CTAB DNA extraction method more efficient than DNA extraction kits?

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Comparison of DNA extraction methods for non-marine molluscs: is modified CTAB DNA extraction method more efficient than DNA extraction kits?

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