Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGE

The PCR internal standard (PCRIS) originates from the Drosophila melanogaster genome and is a 140-basepair long PCR product, which is amplified by non-competitive primers in the same PCR reaction tubes as the target DNA and analysed together with the target PCR product on the same DGGE gel. The use of PCRIS during PCR significantly reduced variation among replicate samples both when assessing total PCR product and when comparing bands representing species on a DGGE gel. The entire ISMAD protocol was shown to accurately describe changes in relative abundance in an environmental sample using PCR-DGGE. It should, however, be mentioned that despite the use of ISMAD some inherent biases still exist in DNA extraction and PCR-DGGE and these should be taken into consideration when interpreting the diversity in a sample based on a DGGE gel.