PCR-based approach for sequencing mitochondrial genomes of decapod crustaceans, with a practical example from kuruma prawn (Marsupenaeus japonicus).
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.
Yamauchi
Mitsugu M
MM
Ocean Research Institute, University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan. ymm@ori.u-tokyo.ac.jp
Miya
Masaki U
MU
Machida
Ryuji J
RJ
Nishida
Mutsumi
M
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D017931:DNA Primers; D033364:Decapoda / Q000235:genetics*; D016678:Genome; D008928:Mitochondria / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers