High-Throughput Direct DNA Analysis from Intact Fish Eggs By Real-Time PCR

Current protocols to analyze DNA from fish eggs require multi-step DNA extraction prior to analysis; this limits throughput and increases the sample-to-results time. To overcome these limitations, we developed a workflow that can quantitatively analyze the DNA in a single intact fish egg directly by real-time PCR. Commercial Mallotus Villosus (Capelin) eggs were washed, added individually to wells of a PCR plate and analyzed using an inhibitor-tolerant real-time SYBR Green qPCR supermix and target-specific primers. We find that mitochondrial cytochrome b (Cytb) DNA can be amplified efficiently and specifically from a single egg with over 95% success. Our finding demonstrates that sample preparation can be bypassed entirely for analysis of fish egg DNA if inhibitor-tolerant real-time PCR reagents are used. This rapid, easy and inexpensive approach of analyzing fish egg DNA can benefit aquaculture breeding and stock identification. It is also plausible that this methodology can extend to other fields that can benefit from direct analysis of DNA from crude samples.