PCR-based approach for sequencing mitochondrial genomes of decapod crustaceans, with a practical example from kuruma prawn (Marsupenaeus japonicus)

• Published in: Marine biotechnology (New York, N.Y.) Vol. 6; no. 5; pp. 419 - 429
• Main Authors: Yamauchi, Mitsugu M, Miya, Masaki U, Machida, Ryuji J, Nishida, Mutsumi
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01.10.2004
• Subjects: Animals; Base Sequence; Chromosome Mapping; Crustaceans; Decapoda; Decapoda (Crustacea) - genetics; DNA Primers; Genome; Genomes; Marine; Marsupenaeus japonicus; Microbiology; Mitochondria - genetics; Mitochondrial DNA; Molecular Sequence Data; Polymerase Chain Reaction - methods; Sequence Alignment; Sequence Analysis, DNA - methods
• ISSN: 1436-2228; 1436-2236
• DOI: 10.1007/s10126-003-0036-2

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.