Origin of spurious multiple bands in the amplification of microsatellite sequences

Multiple band artifacts are seen commonly in the analyses of short repetitive sequences, also known as microsatellites, using the polymerase chain reaction (PCR). In this study, the conditions of PCR were examined for five microsatellite loci (D2S119, D2S123, D5S409, D11S904, and interferon alpha) in an attempt to eliminate this artifact. In addition, and a possible mechanism for the formation of the multiple band artifact in non-denaturing polyacrylamide gel electrophoresis was also explored. The intensity of multiple bands increased when the numbers of PCR cycles were increased. The multiple bands were abolished simply by reducing PCR cycle numbers and were reproduced from single specific PCR products undergoing alternate denaturation and reassociation without primer extension. This finding suggests that formation of multiple bands in non-denaturing gel electrophoresis is a result of improper annealing of PCR fragments, rather than being the result of polymerase slippage and 3' non-template extension, as has been reported previously.