2.5 | Primer design
Beal et al . (2019) reported a correlation between methylation
rate and age in the common bottlenose dolphin (a closely related species
to the Indo-Pacific bottlenose dolphin) using three gene regions:TET2 , GRIA2 , and CDKN2A from DNA extracted from
skin samples. Similar correlations using the same genes were seen in
humpback whales (Polanowski et al ., 2014) and Antarctic minke
whales (Tanabe et al., 2020). These findings suggest that these
genes can be commonly used for epigenetic clock analyses in cetaceans.
However, we were unable to find the genomic information on Indo-Pacific
bottlenose dolphins. Therefore, primer designs were referred to the
genomic information on common bottlenose dolphins, available at the
National Centerfor Biotechnology Information (NCBI,https://www.ncbi.nlm.nih.gov/)
database using the Standard Nucleotide Basic Local Alignment Search Tool
(BLAST) (Altschul et al ., 1990). We attempted to design PCR
primers to amplify three target genes (reference genomes: TET2 &GRIA2 : NC_047038; CDKN2A : NC_04739). Primers were
designed using Methyl Primer Express v1.0 (Thermo Fisher Scientific, San
Jose, CA, USA, Table 1). However, we were unable to design a primer for
the TET2 gene that would successfully amplify the target region.
As a result, we proceeded with the analysis using only GRIA2 andCDKN2A genes.
There was a need for additional verification to ensure that the designed
primers specifically amplified the DNA of the target species and not the
prey species. To confirm this, we used the NCBI database using the
BLASTN tool
(https://blast.ncbi.nlm.nih.gov/Blast.cgi)
and homogenous gene regions were searched against reference genomes to
ensure that the same sequences were not found in the prey species for
this population, as reported by Takahashi et al . (2020) and Kitaet al. (2018).