Effect of single mismatches at 3'-end of primers on polymerase chain reaction

Objective and method: To investigate the effect of three different mismatches (G/T, G/A or G/G) at the 3'-end of a primer to amplify a 268 bp (base pair) region of the human β-globin gene using different annealing temperatures (45 to 65°C).

Results: The primer with the G/T mismatch was as efficient as the normal primer (G/C match) in the amplification of a 268 bp product at all temperatures tested. However, the primers having G/A or G/G mismatches at the 3'-end did not produce any specific polymerase chain reaction (PCR) fragment at all the annealing temperatures used, except a barely detectable 268 bp product for the G/G mismatch at 45 and 50°C.

Conclusion: We conclude that our PCR system was refractory to amplification when one of the primers contained a G/A or G/G mismatch at the 3'-end with template DNA.