Short Communication Universal primers to amplify the complete mitochondrial 12S rRNA gene in marine fish species

Abstract

ABSTRACT.A pair of new universal 12S mitochondrial rRNA gene primers was designed through multiple alignment analysis of the mitochondrial tRNA Phe and the 5' region of 16S mitochondrial rRNA genes of different kinds of fishes. The primers were successfully used to amplify an expected product fragment of about 1.2 kb from various marine fish species, and the amplified DNA fragment was recognized to contain the complete 12S mitochondrial rRNA and tRNAVal genes, as well as a partial 16S mitochondrial rRNA gene of about 146 bp in length. The primers would facilitate the study of the species discrimination, population and evolution in marine fish species.

An automated workflow to assess completeness and curate GenBank for environmental DNA metabarcoding: The marine fish assemblage as case study

“…For each sample, genomic DNA was extracted from muscle tissue or fin using the Wizard Genomic DNA Purification kit (Promega) following manufacturer's instructions for “Isolating Genomic DNA from Tissue Culture Cells and Animal Tissue.” Extracted DNA was resuspended in Milli‐Q water and its concentration was determined with NANODROP (Thermo Scientific™). The extracted DNA was then amplified using the MarineFish primer pair (Jin et al, 2013), a 900‐ to 1100‐bp‐long 12S rRNA region covering both teleo and MiFish regions, by mixing 10 μL of 2X PCR Master Mix (Fisher Scientific), 0.4 μL of each primer, 2 μL DNA template (1–20 ng), and 7.2 μL of nuclease‐free water, and using the following amplification conditions: 95°C for 3 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 75 s; and final extension at 72°C for 10 min. The PCR products were migrated in a 2% agarose gel in TAE buffer and purified using ILUSTRA EXOSTAR1‐Step (Cytiva) following manufacturer's conditions and sent for Sanger sequencing.…”

“…Extracted DNA was resuspended in Milli-Q water and its concentration was determined with NANODROP (Thermo Scientific™). The extracted DNA was then amplified using the MarineFish primer pair (Jin et al, 2013), a 900-to 1100-bp-long 12S rRNA region covering both teleo and MiFish regions, by mixing 10 μL of 2X PCR Master Mix (Fisher Scientific), 0.4 μL of each primer, 2 μL DNA template (1-20 ng), and 7.2 μL of nuclease-free water, and using the following amplification conditions: 95°C for 3 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 75 s; and final extension at 72°C for 10 min.…”

An automated workflow to assess completeness and curate GenBank for eDNA metabarcoding: the marine fish assemblage as case study

“…Extracted DNA was resuspended in Milli-Q water and its concentration was determined with NANODROP (Thermo Scientific™). The extracted DNA was then amplified using the MarineFish primer pair (Jin, Zhao et al 2013), a 900-1,100 bp-long 12S rRNA region covering both teleo and MiFish regions, by mixing 10 μL of 2X PCR Master Mix (Fisher Scientific), 0.4 μL of each primer, 2 μL DNA template (1-20 ng), and 7.2 μL of Nuclease Free water, and using the following amplification conditions: 95°C for 3 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 75 s; and final extension at 72°C for 10 min. The PCR products were migrated in a 2% agarose gel in TAE buffer and purified using ILUSTRA EXOSTAR1-Step (Cytiva) following manufacturer´s conditions and sent for Sanger sequencing.…”