Peptide nucleic acid-mediated PCR clamping as a useful supplement in the determination of microbial diversity

Peptide nucleic acid (PNA)-mediated PCR clamping (H, Orum, P, E. Nielsen, M , Egholm, R, H, Berg, O, Buchardt, and C, Stanley, Nucleic Acids Res. 21:53 32-5336, 1993) was introduced as a novel procedure to selectively amplify r ibosomal DNAs (rDNAs) which are not frequently found in clone libraries gen erated by standard PCR from complex microbial consortia. Three different PN A molecules were used; two of these molecules (PNA-ALF and PNA-EUB353) over lapped with one of the amplification primers, whereas PNA-1114F hybridized to the middle of the amplified region. Thus, PCR clamping was achieved eith er by competitive binding between the PNA molecules and the forward or reve rse primers (competitive clamping) or by hindering polymerase readthrough ( elongation arrest). Gene libraries generated from mixed rDNA templates by u sing PCR clamping are enriched for clones that do not contain sequences hom ologous to the appropriate PNA oligomer, This effect of PCR clamping was ex ploited in the following two ways: (i) analysis of gene libraries generated by PCR clamping,vith PNA-ALF together with standard libraries reduced the number of clones which had to be analyzed to detect all of the different se quences present in an artificial rDNA mixture; and (ii) PCR clamping with P NA-EUB353 and PNA-1114F was used to selectively recover rDNA sequences whic h represented recently described phylogenetic groups (NKB19, TM6, cluster r elated to green nonsulfur bacteria) from an anaerobic, dechlorinating conso rtium described previously. We concluded that PCR clamping might be a usefu l supplement to standard PCR amplification in rDNA-based studies of microbi al diversity and could be used to selectively recover members of undescribe d phylogenetic clusters from complex microbial communities.