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

ABSTRACT

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 beta-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