Detection of genetically modified coho salmon using polymerase chain reaction (PCR) amplification.
A PCR-based protocol for the identification of genetically modified salmon carrying a growth hormone transgene was developed. Several primer pairs were examined, and the primers that gave consistent results were selected to conduct routine testing. Comparison among several DNA extraction procedures, as well as different buffer compositions, led to the adoption of TriZol as the method of choice. Low potassium and high magnesium chloride concentrations were very important in the overall success of the PCR reaction, whereas buffer pH, ranging from 8.3 to 9.2, had little impact on the amplification reaction. The optimal primer annealing temperature was 52 degrees C. Although fish muscle tissues were the primary source for DNA samples, detection of the transgene was also possible in bones, skin, fins, and other organs. No benefits were achieved by the addition of additives such as dimethyl sulfoxide and betaine to the PCR reaction. This optimized PCR method was used to identify all samples tested (61 samples and 17 controls) with 100% accuracy.
Masri
Saad
S
CFIA, Centre for Plant Health, 8801 East Saanich Road, Sidney, British Columbia V8L 1H3, Canada. masris@inspection.gc.ca
Rast
Heidi
H
Ripley
Teresa
T
James
Delano
D
Green
Margaret
M
Jia
Xiaoyan
X
Devlin
Robert H
RH
D000818:Animals; D030801:Animals, Genetically Modified; D002021:Buffers; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D004247:DNA / Q000032:analysis; D030841:Food, Genetically Modified; D013006:Growth Hormone / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000008:administration & dosage; D018482:Muscle, Skeletal / Q000737:chemistry; D017684:Oncorhynchus kisutch / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011188:Potassium / Q000008:administration & dosage; D013696:Temperature
D002021:Buffers; D002593:Cetrimonium Compounds; D015636:Magnesium Chloride; D013006:Growth Hormone; D004247:DNA; D011188:Potassium; D000077286:Cetrimonium