DNA extraction from recently fertilised Atlantic salmon embryos for use in microsatellite validation of triploidy
Callum Howard, John B. Taggart, Caroline R. Bradley, Alejandro P. Gutierrez, John F. Taylor, Paulo A. Prodöhl, Herve Migaud, Michaël Bekaert
DNA extraction
DNA quality and quantity assessments
Microsatellites validation assessment
Egg
Tiploid
Salmon
Abstract
The current methods used for producing triploid Atlantic salmon are generally reliable but not infallible, and each batch of triploids must be validated to ensure consumer trust and licensing compliance. Microsatellites have recently been shown to offer a cheaper and more convenient alternative to traditional flow cytometry for triploidy validation in a commercial setting. However, incubating eggs to at least the eyed stage for microsatellite validation poses challenges, such as reduced quality and performance of triploids produced from later eggs in the stripping season. To address these issues, we propose another option: extracting DNA from recently fertilised eggs for use in conjunction with microsatellite validation. To achieve this, we have developed an optimized protocol for HotSHOT extraction that can rapidly and cheaply extract DNA from Atlantic salmon embryos, which can then be used for triploidy validation through microsatellites. Our approach offers a simpler and more cost-effective way to validate triploidy, without the need for skilled dissection or expensive kits.
Steps
DNA extraction
If eggs stored in ethanol, remove using forceps and place on clean tissue to remove excess ethanol.
Place embryos in a beaker of Tris-HCl (5millimolar (mM), 8) for 0h 15m 0s.
Remove the eggs and remove excess liquid with clean tissue.
For low throughput needs the eggs can then be placed into individual 1.5 mL screw cap tubes, for high throughput needs the eggs can be places, one per well, into a 2 mL deep 96-well plate.
Pierce the chorion by applying pressure using the end of the forceps.
Add 400µL alkaline lysis buffer to each tube/well and seal.
Invert 5 times, and placed into either a heat block or a laboratory oven running at 90°C for 0h 30m 0s.
Remove and place On ice for 0h 5m 0s.
Unseal and add an equal amount (400µL) of neutralisation buffer.
Reseal and rapidly invert 10 times and then spin down briefly using a centrifuge.
Spin down for 14000rpm (or 20,000 g).
Collect the middle layer of the solution.
The DNA (middle layer) can now be used instantly, stored at 4°C for up to a week, or stored at -18°C for use later on.
DNA quality and quantity assessments
0.5µL of sample DNA (middle layer), 3µL MyTaq HS mix (Bioline, USA), 0.6picomolar (pM) of each primer (0.12µL) and 2.26µL ultrapure water in PCR tube or plate (10µL total). Perform PCR at the appropriate thermal cycle for gene of interest.
In this case, 38 cycles of
95°C for 0h 0m 15s,
60°C for 0h 0m 15s and
72°C for 0h 0m 40s.
Load 2.5µL of the PCR product into a 1.25% agarose gel with 5µL of 1.5× loading dye (ThermoFisher Scientific, UK) in 0.5× TAE buffer.
Migrate the gel with ethidium bromide and visualised under UV in a transilluminator for the quality of bands and the presence of smear or primer dimer.
1µL of sample DNA (middle layer), 5µL Sensifast SYBR No-ROX kit (Bioline, USA), 1picomolar (pM) of each primer (0.2µL), 3.6µL ultrapure water in qPCR plate. Perform qPCR starting by 95°C for 0h 3m 0s followed by the appropriate thermal cycle for gene of interest.
In this case, 40 cycles of
95°C for 0h 0m 15s,
60°C for 0h 0m 15s and
72°C for 0h 0m 30s.
Microsatellites validation assessment
30µL of sample loading solution (SLS), and 0.35µL of size standard (WellRED size standard, Eurofins, Germany) and add to well of capillary electrophoresis plate. Top each well off with one drop of mineral oil.
Run capillary electrophoresis machine (Beckman Coulter CEQ 8000, Beckman Coulter, USA) according to the manufacturer’s instructions.
