Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA

Abstract

We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a variety of bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.

Until recently, the estimation of bacterial biodiversity has been hampered by limitations associated with cultivating bacteria from natural environments. An uncultured fraction has been recognized to be a major component of all microbial communities (7). The application of molecular approaches to the characterization of bacterial communities has overcome the requirement for prior cultivation of community members. In particular, the analysis of 16S rRNA genes, aided by using PCR to amplify target sequences in environmental samples, has enabled molecular ecologists to provide better estimates of bacterial diversity (1). PCR primers for amplification of 16S rRNA genes are widely available (6, 13). However, a problem with several commonly used amplimers is that they have been constructed theoretically, using the (incomplete) database of 16S rRNA sequences from cultured organisms, and have not been tested systematically. Hence, empirical testing is essential to confirm PCR primer specificity prior to their use in PCRs with environmental samples.

Primers for PCR described by Lane (6), which have been used by many other workers (5, 8), consistently failed to work with some difficult samples generated in our work. DNA samples extracted from various sources, including deep sea sediment, oral bacteria, and bacteria isolated from epilithon (biofilms associated with stones in lotic habitats), were found to be poor templates for amplification of the 16S rRNA gene with amplimers such as 27f and either 1492r or 1392r (numbering is based on the Escherichia coli 16S rRNA gene [3]). We had attempted to optimize the PCR conditions for 27f-1392r by altering the annealing temperature, Mg²⁺ concentration, and DNA template concentration and also by including the PCR additives bovine serum albumin, Triton X-100, T4 gene 32 protein, polyethylene glycol 8000 and glycerol. Finally, we decided to redesign the amplimers, and when we used 63f and 1387r with the difficult DNA samples, we were successful.

The wide adoption of amplimers 27f, 1392r, and 1492r is empirically based, and although their utility for investigating the molecular ecology of natural bacterial communities is often assumed, to our knowledge they have not been systematically tested. In this communication, we describe a new set of amplimers which were designed to be universal for the domain Bacteria (14) and their testing on a range of pure cultures and difficult natural samples.

Unless otherwise noted, genomic DNA was extracted from pure cultures, reference strains, and environmental samples by a modification of the method of Ausubel et al. (2). DNA from marine sediment samples (1 to 2 g), collected as described previously (9), was extracted by a modification of the method of Rochelle et al. (10). After the lysozyme step, 100 μl of proteinase K (18 mg/ml; Sigma) was added, and the solution was further incubated for 1 h at 37°C. Second, the phenol-chloroform step was replaced by adding one-half volume of 7.5 M ammonium acetate, and the mixture was centrifuged at 11,220 × g_av for 20 min at 4°C. DNA from the three Leptospira strains was obtained as a thermolysate (2a).

Two PCR primers were designed (Oligo, version 3.4; National Biosciences Inc.) to amplify approximately 1,300 bp of a consensus 16S rRNA gene (6): forward primer 63f (5′-CAG GCC TAA CAC ATG CAA GTC-3′) and reverse primer 1387r (5′-GGG CGG WGT GTA CAA GGC-3′) (Pharmacia). Primers 27f and 1392r (6) were also used.

The PCR mixtures (100 μl) contained 20 pmol of each appropriate primer, 200 μM each deoxynucleoside triphosphate, Taq extender PCR buffer (Stratagene Ltd.), 0.5 U of Taq extender (Stratagene), and 0.5 U of Taq polymerase (Boehringer). Approximately 200 to 300 ng of DNA from a test strain culture and subnanogram quantities of sediment DNA were added to PCRs. In addition, 2 μg of T4 gene 32 protein (Pharmacia) was included in PCRs of sediment DNA. PCR was performed with one of the following thermal cyclers: Hybaid Omnigene, Omni-E, or TR1; Perkin-Elmer 460; or MJ Research PTC-100. All cyclers were programmed to perform 30 cycles consisting of 95°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min followed by a final extension step of 5 min at 72°C. PCR products were visualized by agarose gel electrophoresis (11).

The specificities of the new primers were tested in PCRs with template DNA from cultures of well-characterized species representing the major groups of the domain Bacteria (Table 1) (14). Since DNA from the Thermotogales, green non-sulfur bacteria, and Fibrobacteria groups was not available for testing, the sequence similarities between amplimers 63f and 1387r and the 16S rRNA genes of selected species from those groups were assessed by aligning the amplimer sequences with the appropriate prealigned sequences obtained from the Antwerp rRNA database (12) (Table 2). In the comparison of alignments shown in Tables 2 and 3, one main point emerged. Amplimers 63f and 1387r successfully amplified 16S rRNA genes from species showing higher levels of theoretical 5′ mismatches than amplimer pair 27f-1392r, in some cases with 3′ mismatches in 1387r.

TABLE 1.

Species of bacteria and archaea tested in PCRs using amplimer 63f and 1387f to amplify 16S rRNA genes

Straina	Woese grouping	PCR amplificationb
Chlamydia psittacic	Planctomyces and relatives	+
Synechocystis sp. strain PCC 6803	Cyanobacteria and chloroplasts	+
Synechococcus sp. strain WH 7803	Cyanobacteria and chloroplasts	+
Synechococcus sp. strain PCC 7942	Cyanobacteria and chloroplasts	+
Nostoc sp.	Cyanobacteria and chloroplasts	+
Anabaena sp. strain PCC 7120c	Cyanobacteria and chloroplasts	+
Runella sp.	Flexibacter-Cytophaga-Bacteroides phylum	+
Prevotella oris	Flexibacter-Cytophaga-Bacteroides phylum	+
Prevotella veroralis	Flexibacter-Cytophaga-Bacteroides phylum	+
Prevotella buccalis	Flexibacter-Cytophaga-Bacteroides phylum	+
Prevotella intermedia	Flexibacter-Cytophaga-Bacteroides phylum	+
Cytophaga ochracea	Flexibacter-Cytophaga-Bacteroides phylum	+
Rhodobacter sphaeroides	α-Proteobacteria	+
Alcaligenes eutrophus JMP222	β-Proteobacteria	+
Myxococcus xanthus NCIMB 9412c	δ-Proteobacteria	+
Desulfovibrio desulfuricansc	δ-Proteobacteria	+
Desulfovibrio salexigensc	δ-Proteobacteria	+
Desulfovibrio sapovorans	δ-Proteobacteria	+
Desulfobacter postgatei	δ-Proteobacteria	+
Desulfobulbus propionicusc	δ-Proteobacteria	+
Wolinella succinogenes ATCC 29543	ɛ-Proteobacteria	+
Fusobacterium nucleatumc	ɛ-Proteobacteria	+
Campylobacter concisus ATCC 33237	ɛ-Proteobacteria	+
Campylobacter sputorum subsp. sputorum ATCC 35980	ɛ-Proteobacteria	+
Campylobacter rectus ATCC 33238	ɛ-Proteobacteria	+
Campylobacter curvus ATCC 35224	ɛ-Proteobacteria	+
Campylobacter showae ATCC 51146	ɛ-Proteobacteria	+
Escherichia coli HB101c	γ-Proteobacteria	+
Acinetobacter calcoaceticus AC8250	γ-Proteobacteria	+
Pseudomonas putida UWC1c	γ-Proteobacteria	+
Klebsiella pneumoniae	γ-Proteobacteria	+
Eubacterium nodatum ATCC 33099c	Clostridia and relatives	+
Eubacterium brachy ATCC 33089	Clostridia and relatives	+
Eubacterium timidum ATCC 33092, ATCC 33093	Clostridia and relatives	+
Eubacterium tardum SC68,c SC88P	Clostridia and relatives	+
Eubacterium exiguum ATCC 700122	Clostridia and relatives	+
Eubacterium minutum ATCC 700079, ATCC 700080	Clostridia and relatives	+
Eubacterium saburreum ATCC 33271, ATCC 33318, ATCC 33319, ATCC 43850	Clostridia and relatives	+
Pseudoramibacter alactolyticus DSM 3980 (= ATCC 23263)	Clostridia and relatives	+
Ruminococcus torques ATCC 27756	Clostridia and relatives	+
Veillonella atypica ATCC 17744	Clostridia and relatives	+
Veillonella dispar ATCC 17748	Clostridia and relatives	+
Veillonella parvula ATCC 10790	Clostridia and relatives	+
Veillonella caviae ATCC 33540	Clostridia and relatives	+
Veillonella criceti ATCC 17747	Clostridia and relatives	+
Veillonella rodentium ATCC 17743	Clostridia and relatives	+
Veillonella ratti ATCC 17746	Clostridia and relatives	+
Eubacterium lentum NCTC 11813,c ATCC 25559	Gram positive, high G+C	+
Micrococcus luteus	Gram positive, high G+C	+
Actinomyces naeslundii genosp. 1 ATCC 12104	Gram positive, high G+C	+
Actinomyces naeslundii genosp. 2 WVU 627	Gram positive, high G+C	+
Actinomyces israelii ATCC 12102, ATCC 10048, MTU 01003, MTU 01004	Gram positive, high G+C	+
Actinomyces gerencseriae ATCC 23860	Gram positive, high G+C	+
Actinomyces odontolyticus ATCC 17929	Gram positive, high G+C	+
Actinomyces meyeri ATCC 35568	Gram positive, high G+C	+
Actinomyces georgiae ATCC 49285	Gram positive, high G+C	+
Actinomyces viscosus ATCC 15987	Gram positive, high G+C	+
Propionibacterium acnes ATCC 11827	Gram positive, high G+C	+
Bacillus cereus NCTC 7464	Bacillus-Lactobacillus-Streptococcus subdivision	+
Bacillus popilliae NRRL B2309Mc	Bacillus-Lactobacillus-Streptococcus subdivision	+
Peptostreptococcus anaerobius	Bacillus-Lactobacillus-Streptococcus subdivision	+
Lactobacillus delbrueckii subsp. delbrueckii ATCC 9649	Bacillus-Lactobacillus-Streptococcus subdivision	+
Lactobacillus coryniformis subsp. torquens ATCC 25600	Bacillus-Lactobacillus-Streptococcus subdivision	+
Streptococcus salivarius ATCC 7073	Bacillus-Lactobacillus-Streptococcus subdivision	+
Streptococcus sanguis ATCC 10556	Bacillus-Lactobacillus-Streptococcus subdivision	+
Eubacterium saphenum ATCC 49989	Gram positive, low G+C	+
Leptospira inadai ATCC 43289	Spirochaeta-Treponema-Borrelia subdivision	+d
Leptospira kirschneri ATCC 23469	Spirochaeta-Treponema-Borrelia subdivision	+d
Leptospira wolbachii ATCC 43284	Spirochaeta-Treponema-Borrelia subdivision	+d
Treponema denticola ATCC 35405, ATCC 33520	Spirochaeta-Treponema-Borrelia subdivision	+
Treponema vincentii ATCC 35580	Spirochaeta-Treponema-Borrelia subdivision	+
Treponema pallidum subsp. pallidum Nichols strain	Spirochaeta-Treponema-Borrelia subdivision	+
Treponema phagedenis biovar Kazan ATCC 27087, biovar Reiter	Spirochaeta-Treponema-Borrelia subdivision	+
Treponema socranskii subsp. socranskii ATCC 35536	Spirochaeta-Treponema-Borrelia subdivision	+
Treponema medium G7201	Spirochaeta-Treponema-Borrelia subdivision	+
Methanosarcina frisius	Euryarchaeota, Methanosarcinale	−
Halobacterium halobium NCMB 2090	Euryarchaeota, extreme halophile	−

^aStrains not identified as originating from specific culture collections were well-characterized strains from culture collections held by participating laboratories. 

^b+, positive reaction, i.e., a single amplicon of the correct predicted size (ca. 1,300 bp); −, no PCR product. 

^cThe PCR product of this strain was sequenced to ensure that it represented the 16S rRNA gene from the organism. 

^dThe 16S rRNA gene from this Leptospira species was successfully amplified; however, the resulting amount was approximately 90% less than the signal generated an amplification of the 16S rRNA gene of E. coli. 

TABLE 2.

Theoretical alignment of sequences of amplimers 63f and 1387r with database sequences of 16S rRNA genes from species not tested by PCR

Strain	Woese grouping	Sequence vsa:
		63f	1387r
		CAGGCCTAACACATGCA—AGTC	CGGAACATGTGWGGCGGG
Fibrobacter intestinales	Fibrobacter spp.	AGTGTCTTATACATGCA—AGTC	GCCTTGTACACACCGCCC
Chlorobium limicola	Green non-sulfur	CGTGCCTAACACATGCA—AGTC	GCCTTGTACACACCGCCC
Chloroflexus aurantiacus	Green non-sulfur	CGTGCCTAATGCATGCA—AGTC	GCCTTGTACACACCGCCC
Herpetosiphon aurantiacus	Green non-sulfur	CGTGCCTAATGCATGCA—AGTC	GCCTTGTACACACCGCCC
Thermomicrobium roseum	Green non-sulfur	CGTGCCTAATGCATGCA—AGTC	GCCTTGTACACACCGCCC
Deinococcus radiodurans	Radioresistant micrococci and relatives	CGTGCTTAAGACATGCA—AGTC	GCCTTGTACACACCGCCC
Thermus aquaticus	Radioresistant micrococci and relatives	CGTGCCTAAGACATGCA—AGTC	GCCTTGTACACACCGCCC
Thermus ruber	Radioresistant micrococci and relatives	TATGCCTAAGACATGCA—AGTC	GCCTTGTACACACCGCCC
Fervidobacterium nodosum	Thermotogales	CGTGCCTAACACATGCA—AGTC	GCCTTGTACACACCGCCC
Thermotoga elfii	Thermotogales	CGTGCTTAACACATGCA—AGTC	GGTTTGCACACACCGCCC
Thermotoga maritima	Thermotogales	CGTGCCTAACACATGCA—AGTC	GCCTTGTACACACCGCCC

^aNucleotides underlined and in boldface differ from corresponding nucleotides in amplimer. 

TABLE 3.

Alignment of sequences of amplimers 63f-1387r and 27f-1392r with the corresponding regions of 16S rDNA genes of representative species used in PCR

Strain	Woese group	Sequence vs primersa:
		27f and 63f	1387r and 1392r
		AGAGTTTGATCMTGGCTCb            CAGGCCTAACACATGCAA—GTCc	CRTGTGTGGCGGGCAd
			CGGAACATGTGMGGCGGGe
Rhodobacter sphaeroides ATH 2.4.1	α-Proteobacteria	GAGAGTTTGATCCTGGCTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Alcaligenes eutrophus	β-Proteobacteria	AAGAGTTTGATCCTGGCTCAGATTnAACGCTGGCGGCATGCCTTACACATGCAA—GTC	GTCTTGTACACACCGCCCGT
Campylobacter concisus (FDC 288)	ɛ-Proteobacteria	GAGAGTTTGATCCTGGCTCAGAGTGAACGCTGGTGGCGTGCCTAATACATGCAA—GTC	GTCTTGTACTCACCGCCCGT
Campylobacter curvus (ATCC 35224T; VPI 9584)	ɛ-Proteobacteria	GAGAGTTTGATCCTGGCTCAGAGTGAACGCTGGCGGCGTGCCTAATACATGCAA—GTC	GTCTTGTACTCACNGCCCGT
Campylobacter rectus (CCUG 19168)	ɛ-Proteobacteria	GAGAGTTTGATCCTGGCTCAGAGTGAACGCTGGCGGCGTGCCTAATACATGCAA—GTC	GTCTTGTACTCACCGCCCGT
Campylobacter showae (CCUG 3054)	ɛ-Proteobacteria	GAGAGTTTGATCCTGGCTCAGAGTGAACGCTGGCGGCGTGCCTAATACATGCAA—GTC	GTCTTGTACTCACCGCCCGT
Desulfobacter postgatei	δ-Proteobacteria	GAGAGTTTGATCCTGGCTCAGAATGAACGCTGGCGGCGTGCTTAACACATGCAA—GTC	GNNTTGTACACACCGCCNGT
Desulfovibrio desulfuricans (ATCC 27774)	δ-Proteobacteria	GAGAGTNTGATTCTGGCTCAGATTGAACGCTGGCGGCGTGCTTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Myxococcus xanthus DK1622 (ATCC null)	δ-Proteobacteria	GAGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCGTGCCTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Acinetobacter calcoaceticus	γ-Proteobacteria	AAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Escherichia coli	γ-Proteobacteria	AAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAA—GTC	CCTTGTACACACCGCCCGTC
Pseudoramibacter alactolyticus DSM 3980, ATCC 23263	γ-Proteobacteria	-AGAGTTTGATYMTGGCTCAGGACGAACGCTGGCGGTATGCTTAACACATGCAA—GTC	GTCTTGTACACACCGCCCGT
Bacillus cereus F4810/72 serotype 1 (NCTC 11143)	Bacillus-Lactobacillus-Streptococcus subdivision	GAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Bacillus popilliae	Bacillus-Lactobacillus-Streptococcus subdivision	GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAAGACATGCAA—GTC	GTCTTGTACACACCGCCCGT
Lactobacillus coryniformis	Bacillus-Lactobacillus-Streptococcus subdivision	GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAA—GTC	GCCTTGTACACACCGCNCGT
Lactobacillus delbrueckii subsp. delbrueckii strain Calvert	Bacillus-Lactobacillus-Streptococcus subdivision	GAGAGTTNGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Streptococcus salivarius C699 (ATCC 13419)	Bacillus-Lactobacillus-Streptococcus subdivision	GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAA—GTA	GCNTNGTACACACCGCCCGT
Streptococcus sanguis (NCTC 7863; ATCC 15300)	Bacillus-Lactobacillus-Streptococcus subdivision	GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATNCNTGCAA—GTN	GCCTTGTACACACCGCCCGT
Eubacterium angustum	Clostridia and relatives	GAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAA—GTC	GTCTTGTACACACCGCCCGT
Eubacterium fossor	Clostridia and relatives	GAGAGTTCGATCCTGGCTCAGGATGAACGCTGGCGGCGCGCCTAACACATGCAA—GTC	GCNTTGTACACACCGCCCGT
Eubacterium lentum	Clostridia and relatives	-AGAGTTTGATNNTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Eubacterium limosum	Clostridia and relatives	GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGTATGCTTAACACATGCAA—GTC	GTCTTGTACACACCGCCCGT
Eubacterium xylanophilum	Clostridia and relatives	GAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAA—GTC	GTCTTGTACACACCGCNCGT
Nostoc muscorum	Cyanobacteria and chloroplasts	GAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGTATGCTTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Prevotella buccalis	Flexibacter-Cytophaga-Bacteroides phylum	TAGAGTTTGATCCTGGCTCAGGATNAACGCTAGCTACAGGCTTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Prevotella intermedia	Flexibacter-Cytophaga-Bacteroides phylum	GAGAGTTTGATCCTGGCTCAGGATNAACGCTAGCTATAGGCTTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Prevotella oris	Flexibacter-Cytophaga-Bacteroides phylum	GAGAGTTTGATCCTGGCTCAGGATNAACGCTGGCTACAGGCTTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Prevotella veroralis	Flexibacter-Cytophaga-Bacteroides phylum	GAGAGTTTGATCCTGGCTCAGGATNAACGCTAGCTACAGGCTTAACACATGCAA—GTC	GCCTTGTACACACCGCCCGT
Actinomyces viscosus	Gram positive, high G+C	GAGAGTTNNATCCTGGCTCAGGACGAANGCTNGCGGCGTGCTTAACACATGCAA—GTC	GGCTTGTACACACCGCCCGT
Propionibacterium acnes	Gram positive, high G+C	GAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAA—GTC	GTCTTGTACACACCGCCCGT
Chlamydia psittaci	Planctomyces and relatives	GAGAATTTGATCTTGGTTCAGATTGAACGCTGGCGGCGTGGATGAGGCATGCAA—GTC	GCCTTGTACACACCGCCCGT
Leptospira inadai ATCC 43289	Spirochaeta-Treponema-Borrelia subdivision	???????????????????????STARCGTTGGCGGCGCGTCTTAAACATGCAA—GTC	ACCTTGTACACACCGCCCGT
Leptospira kirschneri ATCC 23469	Spirochaeta-Treponema-Borrelia subdivision	???????????????????????STARCGCTGGCGGCGCGTCTTAAACATTCCAAGTC	ACCTTGTACACACCGCCCGT
Leptospira wolbachii ATCC 43284	Spirochaeta-Treponema-Borrelia subdivision	???????????????????????STARCGCTGGCGGCGCGTCTTAAACATGCA—ACTC	ACCTTGTACACACCGCCCGT
Treponema denticola	Spirochaeta-Treponema-Borrelia subdivision	GAGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCGCGTCTTAAGCATGCAA—GTC	GCCTTGTACACACCGCCCGT
Treponema phagedenis	Spirochaeta-Treponema-Borrelia subdivision	GAGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCGCGTCTTAAGCATGCAA—GTC	GCCTTGTACACACCGCCCGT

^aPrimer sequences are written 5′→3′. Sequence data on listed species were obtained from the Antwerp ribosomal database. Nucleotides underlined and in boldface differ from corresponding nucleotides in amplimer. 

^bAmplimer 27f sequence. 

^cAmplimer 63f sequence. 

^dAmplimer 1387r sequence. 

^eAmplimer 1392r sequence. 

In a range of experimental studies carried out in the participating laboratories, primers 63f and 1387r were used successfully and consistently to amplify 16S rRNA genes from template DNA extracted from a variety of organisms: organisms identified as belonging to the coryneform and Micrococcus genera (gram-positive, high-G+C bacteria), cultured for the first time from concrete; Eubacterium species cultured from dental abscesses; novel δ-proteobacteria (sulfate- and iron-reducing bacteria); epilithic samples; and deep sea sediments. Conversely, amplimer pair 27f-1392r failed to amplify the 16S rRNA genes of many of these test samples.

Our results provide no clear theoretical explanation for why amplimer pair 63f-1387r was so much more successful than 27f-1392r. One suggestion is that the latter amplimers are not optimal for PCR since 27f may form an intramolecular duplex with a 5′ overhang and may thus be susceptible to the 5′→3′ exonuclease activity of Taq polymerase. Any resultant removal of 5′ nucleotides from 27f (possibly six in total) would affect the annealing temperature of the primer pair (ΔT_m of 13.6°C instead of 1.6°C) and also result in unfavorable intermolecular complementarity between 27f and 1392r, leading to binding of the 3′ ends. An alternative explanation comes from a recent computer analysis of the potential of primers to hybridize with 16S rRNA genes (4). In this study, a primer designed for the conserved area of the 16S rRNA gene, which was also used for 63f, was found to have a greater hybridization potential than the conserved area used for 27f.

In conclusion, although 63f and 1387r showed some theoretical bias, in practice they were more successful than amplimer pair 27f-1392r and amplified 16S rRNA genes from a wider range of bacteria than other primers which are commonly used for bacterial community analysis. So far as we are aware, the other primers have not been tested in the systematic way described for 63f-1387r in this paper. The results presented here suggest that the latter primer pair may be better suited for this type of molecular ecological analysis, the aim of which is to minimize PCR bias, and underline the point that the theoretical design of PCR amplimers is only the beginning and that systematic empirical testing of the amplimers is of paramount importance.