Biased 16S rDNA PCR amplification caused by interference from DNA flanking the template region

Abstract

PCR amplification of 16S rDNA was found to be highly biased, so that the rDNA from one species out of four was preferentially amplified. We present evidence that the observed PCR bias most likely occurs because the genomic DNA of some species contains segments outside the amplified sequence that inhibit the initial PCR steps. Attempts to overcome this bias by use of a ‘touch down’ PCR procedure or by performing PCR in the presence of denaturants or cosolvents such as acetamide, DMSO, or glycerol were unsuccessful. Since the PCR inhibiting interference from template flanking DNA segments evidently is dependent on the position of the primer sites, we suggest that community diversity analysis based on PCR amplification of 16S rDNA can be improved by extending the procedure from comparative analysis of 16S rDNA amplified by use of only one primer set to a procedure involving at least two different 16S rDNA PCR amplifications performed with different primer sets.