Utility of an internal control for the polymerase chain reaction

Abstract

The polymerase chain reaction (PCR) was used to amplify a 209 base-pair fragment of Mycoplasma pneumoniae DNA. The amplicon was transferred into a plasmid and a 680 base-pair piece of foreign DNA was inserted between the two amplimer sites. Plasmid DNA was added to the reaction mixture as an internal control for the polymerase chain reaction. Since the original hybridization target sites were included in this construction, one pair of amplimers could be used to amplify both the target DNA and the internal control DNA. Separation of internal control from target DNA after amplification was easily obtained on agarose gel electrophoresis. For the analysis of clinical samples with the polymerase chain reaction, the addition of internal control DNA allowed monitoring of the overall effectiveness of the amplification in each tube. With this technique approximately one-third of the tests were shown to be unsatisfactory due to technical errors or contaminating inhibitors. Adequate internal controls are necessary to avoid false-negative results with the polymerase chain reaction.