Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions.

Abstract

PCR amplifications of 16S/23S rDNA spacer regions were carried out from conserved 16S and 23S sequences for genomic DNA samples from strains representing 16 bacterial species (12 genera). Multiple products were produced containing conserved homologous sequences at the 3' and S' ends, separated by highly variable internal spacer sequences. These products cross-hybridized forming heteroduplex DNA structures containing double-stranded ends surrounding an internal single-stranded loop. Singlestranded 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 singlestranded 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 decreasing the number of amplification cycles. Heteroduplex and single-stranded DNA structures were also generated by denaturing and reannealing spacer amplification products in the absence of polymerase activity. Whereas heteroduplex and single-stranded DNA structures provide additional information that is helpful in distinguishing between species of bacteria that produce similar homoduplex products, the mobility of heteroduplex and single-stranded DNA structures DNA structures is extremely sensitive to electrophoretic conditions.

PAGE analysis of the heteroduplexes formed between PCR-amplified 16S rRNA genes: estimation of sequence similarity and rDNA complexity

“…Heteroduplexes were usually resolved into two bands that probably correspond to the hybrids formed by each, plus and minus complementary strands of amplified 16s rDNAs. Although the extent of dissimilarity in hybrid pairs is identical, the single-stranded regions in each hybrid may form distinct structural conformations which decrease the mobility to different extents (Jensen & Straus, 1993). The difference in migration between these hybrids is a good indication of the variability that can be observed among heteroduplexes with the same percentage similarity.…”

Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

“…The formation of single-stranded amplicons can be favored by a differential, asymmetric utilization of primers in the PCR amplification due to differences in priming efficiencies, which can result from differences in the GϩC content of the primers used (10). Therefore, primer concentrations were varied at ratios of 1:8 to 8:1 (27f versus 907r; GϩC content, 50 and 37.5%, respectively) to overcome a possible bias related to asymmetric primer utilization in the PCR, but the formation of pseudo-T-RFs was unaffected.…”

“…The formation of partly single-stranded 16S rRNA gene amplicons during PCR may result from template secondary structures (10), which causes the polymerase to pause or fall off the template (23). However, use of the PCR enhancer betaine at various concentrations, which had been shown to be effective in improving the amplification yield and the specificity of templates with high GϩC content or secondary structures (9), did not prevent the formation of pseudo-T-RFs.…”

“…Since restriction endonucleases require double-stranded DNA at the restriction site (21), the presence of singlestranded amplicons in the range of the terminal restriction site was checked for by using mung bean nuclease, which degrades single-stranded DNA (10).…”

Archaeal Diversity of Upland Rice Field Soils Assessed by the Terminal Restriction Fragment Length Polymorphism Method Combined with Real Time Quantitative-PCR and a Clone Library Analysis

“…PCR artifacts can be caused by the formation of a heteroduplex and a chimera. A heteroduplex is formed in PCR by the cross-hybridization of heterologous sequences 9,19,33) , while a chimera is formed from an incompletely extended primer and template switching during multitemplate PCR. Bias can be caused in multitemplate PCR by differences in primer binding energy 8,18,23) and reannealing of templates 19,30) .…”