Direct Amplification of Microsatellite Alleles from Sonicated Goldfish Sperm

Single-locus microsatellite DNA genotyping is now widely used to study parentage and kinship in a variety of animal species (1,3,7) and to identify human individuals (11). Despite the advantage that single-locus DNA amplification can be performed from minute amounts of DNA or even degraded DNA (10), isolation of DNA samples is time-consuming and the material costly to researchers, especially to population geneticists who often deal with a large number of samples. Several simple methods for preparing DNA templates have been reported. For example, formamide low-temperature (FoLT) polymerase chain reaction (PCR) using human blood (9) and direct PCR using microwave-treated hair shafts or blood from mice (8) have been found effective. There are also studies showing that sonication can be used to extract DNA for PCR amplification (2,4,6). We describe an easy and rapid method of amplifying microsatellite alleles directly from sonicated goldfish (Carassius auratus) sperm cells without DNA extraction. Nine unrelated mature male goldfish from a mixed stock (Ozark Fisheries, Stoutland, MO, USA), identified by the presence of operculum tubercles, were stripped of semen (sperm and seminal fluid) after anesthetization with 0.05% 2-phenoxyethanol (Syndel, Vancouver, BC, Canada). A 1-μL semen sample was taken from each of the nine fish and diluted 1:500 with 150 mM NaCl solution (pH 7.2) into a 1.5-mL microcentrifuge tube. The concentration of this sperm dilution was approximately 5 × 107 sperm/mL as measured using a Multisizer II Coulter Counter (Coulter Electronics of Canada, Burlington, ON, Canada). The sperm dilution was sonicated using a Microson Ultrasonic Cell Disruptor (Heat Systems-Ultrasonics, Farmingdale, NY, USA) for 2 min, which caused cell lysis to most of the sperm (checked using a light compound microscope). The sonicated sperm dilution was taken directly for PCR amplification of goldfish microsatellite locus GF1 (12). The sequence of the forward primer was 5′-ATG AAG GGT AGG AAA AGT GTG A-3′, and the reverse primer was 5′-CAG GTT AGG GAG AAG AAG GAA T-3′. The forward primer was labeled with the fluorescent dye TET (PE Applied Biosystems, Foster City, CA, USA). Microsatellite PCR amplification was performed using 1 μL of sonicated sperm dilution in a 25μL reaction volume containing 120 μM each dNTP, 2 mM MgCl2, 0.16 μM each primer, PCR buffer (10 mM TrisHCl, pH 8.8, 0.1% Triton X-100, 50 mM KCl and 0.16 mg/mL bovine