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PCR-Based Approach for Sequencing Mitochondrial Genomes of Decapod Crustaceans, with a Practical Example from Kuruma Prawn (Marsupenaeus japonicus)

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Abstract

An approach for sequencing the entire mitochondrial genomes (mitogenomes) of decapod crustaceans using 79 newly designed and 7 published polymerase chain reaction (PCR) primers is described. The approach comprises the following steps: (1) the entire mitogenome is amplified in 2 or 3 long PCRs; (2) the 86 primers are used in different combinations to amplify contiguous, overlapping short segments of the entire mitogenome with the diluted long PCR products as templates; (3) direct cycle sequencing is conducted using the short PCR products. This strategy allows a more rapid determination of decapod mitogenomic sequences than a traditional method using cloned mitochondrial DNA and primer walking strategy. As a practical example, the mitogenomic sequence for a kuruma prawn Marsupenaeus japonicus (Crustacea: Decapoda), was determined using the PCR-based approach.

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Acknowledgments

We are grateful to T. Komai for confirming the identification of the Marsupenaeus japonicus specimen. We thank graduate students at Molecular Marine Biology Laboratory, Ocean Research Institute, University of Tokyo, for their helpful suggestions during this study. We also thank G.S. Hardy for reading the manuscript. A portion of this study was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (12NP0201 and 13640711).

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Authors and Affiliations

  1. Ocean Research Institute, University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan

    Mitsugu M. Yamauchi, Ryuji J. Machida & Mutsumi Nishida

  2. Department of Zoology, Natural History Museum & Institute, Chiba, 955-2 Aoba-cho, Chuo-ku, Chiba 260-8682, Japan

    Masaki U. Miya

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  1. Mitsugu M. Yamauchi
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  2. Masaki U. Miya
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Correspondence to Mitsugu M. Yamauchi.

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Yamauchi, M.M., Miya, M.U., Machida, R.J. et al. PCR-Based Approach for Sequencing Mitochondrial Genomes of Decapod Crustaceans, with a Practical Example from Kuruma Prawn (Marsupenaeus japonicus). Mar Biotechnol 6, 419–429 (2004). https://doi.org/10.1007/s10126-003-0036-2

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