EFFECT OF PCR CONDITIONS ON THE FORMATION OF HETERODUPLEX AND SINGLE-STRANDED-DNA PRODUCTS IN THE AMPLIFICATION OF BACTERIAL RIBOSOMAL DNA SPACER REGIONS

PCR amplifications of 16S/23S rDNA spacer regions were carried out fro m conserved 16S and 23S sequencer for genomic DNA samples from strains representing 16 bacterial species (12 genera). Multiple products were produced containing conserved homologous sequences at the 3' and 5' e nds, separated by highly variable Internal spacer sequences. These pro ducts cross-hybridized forming heteroduplex DNA structures containing double-stranded ends surrounding an internal single-stranded loop. Sin gle-stranded DNA was also produced in the amplification of rDNA spacer sequences. Fragments comprising the nonhomoduplex DNA components were identified by their susceptibility to removal by digestion with a sin gle-stranded endonuclease. The relative formation of heteroduplex and single-stranded DNA was reduced by reaction conditions favoring primer /template annealing, for example, higher ionic strength, higher primer concentration, and lower annealing temperature, as well as by decreas ing the number of amplification cycles. Heteroduplex and single-strand ed DNA structures were also generated by denaturing and reannealing sp acer amplification products in the absence of polymerase activity. Whe reas heteroduplex and single-stranded DNA structures provide additiona l information that is helpful in distinguishing between species of bac teria that produce similar homoduplex products, the mobility of hetero duplex and single-stranded DNA structures DNA structures is extremely sensitive to electrophoretic conditions.