The effect of primer purity on the efficiency of the amplification of mitochondrial DNA

Primer purity can be a contributing factor in DNA PCR amplification. Synthesized primers which were not purified, some of their counterparts which had been HPLC purified, and primers which were commercially produced for the study were compared using MALDI TOF MS and HPLC with UV detection. Mitochondrial DNA from four individuals was used to assess the comparative efficiency of the three primer sets in PCR amplification. PCR was performed and the products analyzed by CE-UV (Capillary Electrophoresis with UV detection) and HPLC-UV (High Pressure Liquid Chromatography with UV detection) to determine the product size and concentration. The PCR products were also sequenced and their sequences compared to the Anderson Sequence. The sequencing data showed that the un-purified primers generated the smallest number of errors while both the HPLC purified, and the commercially produced primers generated significantly more errors in the PCR reaction.