Myeloid Leukemia

Table 1
Troubleshooting Allele-Specific Polymerase Chain Reaction (PCR)
Problem Possible explanations Suggested solution
No bands Annealing temperature too high Try range of annealing temperatures (54–60ºC)
and choose highest temperature that gives
appropriate results.
Defective reagents or thermal cycler Retry with fresh reagents and, if possible, on
two thermal cyclers.
Control band present but no mutation-specific Annealing temperature too high Try range of annealing temperatures (54–60ºC)
band with V617F-positive DNA and choose highest temperature that gives
appropriate results.
Relative concentration of mutation-specific Try increasing amount of mutation-specific
primer too low primer (up to 5 µL of 10 M primer).
MgCl 2 concentration not optimal Try range of MgCl 2 concentration (1–10
µL of 25 mM solution in a 50-µL reaction).
Mutation-specific band present but no control Homozygous V617F mutation with high No action needed provided V617F-negative
band with V617F-positive DNA clonal burden in granulocytes control DNA gives control band.
Mutation-specific band present in V617F- Annealing temperature too low Try range of annealing temperatures (54–60ºC)
negative DNA and choose highest temperature that gives
appropriate results.
MgCl 2 concentration not optimal Try range of MgCl 2 concentration (1–10 µL of
25 mM solution in a 50-µL reaction).
Contamination with V617F-positive template Retry with fresh reagents, fresh primers,
or PCR product and clean pipets and plasticware.
Distinct bands of the wrong molecular weight Amplification of primer dimers Increase annealing temperature.
Generalized smear of DNA Too much template DNA Reduce amount of DNA per reaction.
Bands in the water control Contamination with V617F-positive Retry with fresh reagents, fresh primers,
template or PCR product and clean pipets and plasticware.
262 Campbell et al.