Every accredited laboratory must validate any procedure, test or equipment it uses in the laboratory. Validation must be completed to ensure the accuracy and robustness of its performance before it can be used paternity testing casework. The validation
process is time consuming and expensive. Non-accredited labs do not have to follow these procedures. This is a typical validation procedure that an accredited laboratory will use when they implement a new STR based paternity testing method.
Primer Pair Titration
Taq DNA Polymerase Titration
Testing Conditions: 0.5X, 0.75X, 1X, 1.5X, 2X primer tested with 30 and 32 cycles
Testing Conditions: 0.5X, 1X, 1.5X, 2X, 4X Taq DNA polymerase tested with 30 and 32 cycles
Magnesium Titration
Comparison of Thermal Cyclers
Testing Conditions: 1mM, 1.25mM, 1.5mM, 1.75mM, 2mM final MgCl2 concentration was tested with 30 and 32 cycles.
Testing Conditions: The models that used are compare to other thermal cyclers in the laboratory.
Variation of Cycle Number
Variation of Annealing Temperature
Testing Conditions: 28, 30, 32, 34 and 36 cycles are tested (10/18, 20, 22, 24 and 26, respectively).
Testing Conditions: 56°C, 58°C, 60°C, 62°C and 64°C annealing temperatures are tested with 30 and 32 cycles.
Variation of Reaction Volume
Testing Conditions: 5µl, 10µl, 12.5µl, 25µl and 50µl reactions are tested with 30 and 32 cycles (template concentration was
held constant with volume changes; 25µl is the recommended reaction volume).
