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).
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Acknowledgements
Authors would like to thank Department of Science and Technology, Government of India (Grant No. EMR_2015/000199/v1/21385) for funding. We would also like to thank Dr. Suzanne Williams, Dr. Praveen Karanth, Dr. Maitreya Sil for help in developing the protocol.
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Chakraborty, S., Saha, A. & Neelavar Ananthram, A. Comparison of DNA extraction methods for non-marine molluscs: is modified CTAB DNA extraction method more efficient than DNA extraction kits?. 3 Biotech 10, 69 (2020). https://doi.org/10.1007/s13205-020-2051-7
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DOI: https://doi.org/10.1007/s13205-020-2051-7