, 1985b), a difference of 2 °C is equivalent to a 6.4% difference in the denaturant. This could yield bands up to 2 cm apart in a 35–65% gel, and multiple bands per 16S rRNA gene sequence could, therefore,

be anticipated. This would invariably lead to multiple bands per 16S rRNA gene sequence, and an overestimation of the diversity. More importantly, the same sequence would yield different banding patterns for different primer batches. The effect of GC-clamp sequence and length variation on band position was then studied experimentally. The V3–5 region of three separate bacterial species of bacteria was amplified using the five sets of primers, and the products were resolved by DGGE. Each lane contained more than one band (Fig. 2a). Importantly, the profiles based on primer sets varied among each other PD-166866 (Fig. 2b). This indicated that DGGE profile variation is due to variation between GC-clamp

primers rather Tacrolimus ic50 than template DNA. One 16S rRNA gene sequence can, therefore, yield multiple bands. The number and distance between the bands appears to be influenced by the specific batch of primers. Three of the five primers (N1–N3) used had an identical sequence design, but displayed deviation both in DGGE patterns and in sequence integrity. DNA sequencing of amplicon pools revealed variation in the GC-clamp sequence, leading to a series of otherwise identical products with different %GC and therefore Tm. Amplicons derived using primer G1 displayed a similar range of variation in GC-clamp sequence and resulting %GC. Primer F1 products displayed the greatest degree of GC-clamp variation and %GC. This during may be due to several adjacent guanosine residues in primer F1. Whether these deviations from the intended sequence occur during synthesis

of the oligonucleotide or during the PCR process is unclear from the current results. Truncation of GC-clamp PCR amplicons of partial 16S rRNA genes has been reported previously (Nubel et al., 1996), and could be due to premature elongation termination of PCR. DNA synthesizers reportedly experience difficulty adding multiple adjacent guanosine residues (Sheffield et al., 1989), and producers of oligonucleotides warn customers of potential problems with the integrity of products with GC-rich stretches. Multiple adjacent guanosine residues reportedly can form aberrant structures such as guanine quartets (Poon & Macgregor, 1998) or four-stranded tetraplexes (Poon & Macgregor, 2000). These structures could interfere during both oligonucleotide synthesis and PCR. Products of primers N1–N3 and F1 lead to a lower degree of GC-clamp variation, and contain only one di-guanosine. Yet, these primers also yielded multiple bands in pure-culture DGGE of all three species, indicating a range of Tm within the amplicon pool. In lieu of multiple guanosines, the GC clamps contained multiple cytosine residues, which would generate multiple guanosines in the reverse strand.