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Applied and Environmental Microbiology, February 2001, p. 880-887, Vol. 67, No. 2
Environmental Sciences Division, Oak Ridge
National Laboratory, Oak Ridge, Tennessee
38831,1 and Center for Microbial
Ecology, Michigan State University, East Lansing, Michigan
488242
Received 8 June 2000/Accepted 30 October 2000
To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and
heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning
approach, a model community of four species was constructed from alpha,
beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR
artifacts were significantly different among the three Taq
DNA polymerases examined: 20% for Z-Taq, with the highest
processitivity; 15% for LA-Taq, with the highest fidelity
and intermediate processitivity; and 7% for the conventionally used
DNA polymerase, AmpliTaq. In contrast to the theoretical
prediction, the frequency of chimeras for both Z-Taq
(8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of
heteroduplexes for Z-Taq were almost three times higher
than those of AmpliTaq. The total PCR artifacts increased
as PCR cycles and template concentrations increased and decreased as
elongation time increased. Generally the frequency of chimeras was
lower than that of mutations but higher than that of heteroduplexes.
The total PCR artifacts as well as the frequency of heteroduplexes
increased as the species diversity increased. PCR artifacts were
significantly reduced by using AmpliTaq and fewer PCR
cycles (fewer than 20 cycles), and the heteroduplexes could be
effectively removed from PCR products prior to cloning by
polyacrylamide gel purification or T7 endonuclease I digestion. Based
upon these results, an optimal approach is proposed to minimize PCR
artifacts in 16S rDNA-based microbial community studies.
The detection, identification, and
characterization of microbial populations and their activities in
environments are a great challenge to microbiologists. The application
of culture-independent nucleic acid techniques has greatly advanced the
detection and identification of microorganisms in natural habitats. In
the last decade, the use of 16S rRNAs or ribosomal DNAs (rDNAs) as
molecular markers has become routine for microbial ecologists. Several
different rRNA-based approaches have been used to characterize
microbial communities, such as cloning plus sequencing
(31), amplified rDNA restriction analysis
(23), terminal restriction fragment length polymorphism
(RFLP) analysis (1, 21), RFLP analysis (25),
denaturing gradient gel electrophoresis (DGGE) (16, 26),
temperature gradient gel electrophoresis (TGGE) (27), single-strand conformation polymorphism analysis (19), and
heteroduplex mobility assay (12). Nearly every study
applying these approaches reveals novel microbial groups, and many of
them are still undetectable by cultivation (2, 3, 7, 8, 11,
13-15, 17, 29, 39, 41). Use of these methods with PCR, however,
can cause bias (32, 33) and artifacts that lead to
overestimation of community diversity (38).
Although PCR-generated chimeras have received much attention (4,
17, 18, 20, 28, 34, 35, 38), PCR-generated heteroduplexes and
mutations have largely been ignored. It is impossible to avoid the
formation of heteroduplexes in the PCR products when a mixture of
homologous genes is used as PCR templates. When a heteroduplex molecule
is cloned and transformed, two homoduplex molecules of 16S rRNA genes
will be produced and segregated as a result of plasmid propagation
(30). When the mixed 16S rRNA genes are subjected to RFLP,
DGGE or TGGE analysis, artificial RFLP patterns or DGGE or TGGE
conformations will be generated.
PCR-generated mutations pose another potential problem. Compared to
other DNA polymerases, Taq DNA polymerase has a higher intrinsic misincorporation rate during synthesis (6). Such errors can accumulate and be enlarged during PCR amplification (38). When an error occurs at the restriction
enzyme-recognizing site, an artificial RFLP pattern will occur.
Although the phenomenon of PCR-induced mutations is well known, their
effects on community diversity studies have not been adequately
addressed. In addition, when the amplified target gene has secondary
structure, deletion mutations may exist (5). Since the 16S
rRNA gene has a stable secondary structure, deletion mutations could be
produced during PCR amplifications.
Theoretically, PCR-generated chimeras should be fewer in amplifications
with DNA polymerases with higher processitivity and decrease as
elongation time increases and cycle number decreases. The PCR-induced
mutations should be lower for DNA polymerases with either higher
fidelity (point mutation) (38) or higher processitivity
(deletion mutation) (5) and decrease as the cycle number
decrease. The frequency for formation of heteroduplexes in the PCR
products should decrease when lesser amounts of PCR products are
synthesized. In addition, there is a potential increase of total PCR
artifacts as species diversity increases. To test these hypotheses and
minimize the three types of PCR artifacts, we evaluated PCR artifacts
under different amplification conditions, and here we provide a general
approach for reducing artifacts in 16S rRNA gene-based cloning studies.
Strains used.
The 16S rRNA genes were amplified from more
than 40 different gram-negative and positive bacteria with the
eubacterium-specific primers FD1 and R1540 (36, 40) and
digested with HhaI. Ten strains that showed distinct
HhaI digestion patterns were selected for constructing model
microbial communities (Table 1).
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.2.880-887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evaluation of PCR-Generated Chimeras, Mutations,
and Heteroduplexes with 16S rRNA Gene-Based Cloning
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and model communities
Experimental design and PCR amplification conditions. The effects of various PCR amplification parameters, including DNA polymerase, cycle number, elongation time, and template concentration, on the formation of PCR artifacts were examined in a four-species community, which contained C1-4, To1-4, B9-12, and P39, one each from the alpha, beta, and gamma subdivisions of the division Proteobacteria, and a gram-positive bacterium. All treatments were carried out in triplicate. For the amplifications above 20 cycles, three amplifications were combined, whereas for the amplifications below 20 cycles, 10 amplifications were combined.
To determine the effect of DNA polymerase on the formation of PCR artifacts, three Taq DNA polymerases were examined: TaKaRa Z-Taq (Pan Vera Corporation, Madison, Wis.), with the highest processitivity; LA-Taq (Pan Vera Corporation), with the highest accuracy and intermediate processitivity; and the conventionally used AmpliTaq DNA polymerase (Perkin-Elmer). PCR amplifications were carried out under the optimum conditions for each Taq as suggested by the manufacturers: in 1× Z-Taq buffer containing 3 mM Mg2+ and a 200 µM concentration of each deoxynucleoside triphosphate (dNTP) with Z-Taq; in 1× GC buffer I, including 2.5 mM Mg2+ and a 400 µM concentration of each dNTP with LA-Taq; and in a buffer of 10 mM Tris-HCl (pH 8.3 at 25°C)-50 mM KCl, containing 1.5 mM Mg2+ and a 200 µM concentration of each dNTP with AmpliTaq. The standard reaction mixture contained 20 pmol of each forward and reverse primer, 230 fg of cloned 16S rDNA (equivalent to 100 pg of genomic DNA) from each strain, bovine serum albumin (100 µg/ml), and 1 U of the Taq DNA polymerase in a final volume of 20 µl. Reaction mixtures were incubated in a thermocycler, (model Gene Amp PCR system 9700; PE Applied Biosystem, Branchburg, N.J.) at 95°C for 5 min, followed by 30 cycles at 95°C for 40 s, 58°C for 30 s, and 72°C for 4 min and then by a final extension at 72°C for 7 min. To examine the effect of elongation time on the formation of PCR artifacts, the 16S rDNAs were amplified with Z-Taq as described above, except the template concentration from each strain was equivalent to 30 ng of genomic DNA. The extension times compared were 20 s, 2 min, and 4 min. To examine the effect of template concentration on the formation of PCR artifacts, the 16S rDNAs were mixed in equal ratios with an equivalent of 100 pg, 1 ng, or 10 ng of genomic DNA per strain and amplified with Z-Taq. In addition, the effect of PCR cycle number on the formation of PCR artifacts was evaluated with Z-Taq for 22, 25, and 28 cycles as well as with AmpliTaq for 15, 20, 25, and 30 cycles. Both amplifications used a mixture of four strains of an equivalent of 100 pg of genomic DNA per strain. To examine the effect of species diversity on the formation of PCR artifacts, model communities were constructed consisting of 4, 7, and 10 species (Table 1). The total template concentration used in each community was equivalent to 500 pg of genomic DNA, with equal ratios of each strain. 16S rDNAs were amplified with AmpliTaq for 15 cycles.16S rRNA gene cloning. Multiple amplications 3 or 10 were combined and purified from low-melting-point agarose gel by using the Wizard PCR Preps DNA Purification System prior to ligation. For amplification with fewer than 20 cycles, PCR products were concentrated by ethanol precipitation. The ratio of 16S rDNAs to pCR II vector was 1:1. Two microliters of each ligation reaction mixture was transformed by heat pulse into E. coli Top10F' competent cells (Invitrogen).
16S rDNA RFLP analysis. The 16S rDNA inserts were amplified directly from transformant cells in 20 µl with primers TA-F and TA-R (41). A 5-µl sample of the amplification mixture was digested with 1 U of HhaI (Gibco BRL, Gaithersburg, Md.) in a final volume of 15 µl at 37°C overnight. The resulting products were resolved by electrophoresis in 1.6% agarose gel in 1× Tris-borate-EDTA (TBE) buffer at 94 V for 4 h. The gel was stained with 0.5 µg of ethidium bromide per ml and visualized by UV excitation. PCR artifacts were detected by comparing the RFLP patterns to those of the reference strains using the Molecular Analyst Program (Bio-Rad, Hercules, Calif.).
Detection of chimeras and mutations. DNA sequencing was carried out on an automated sequencer (model 373A; Applied Biosystems, Foster City, Calif.). Primers FD1 (E. coli 16S rRNA gene position 8 to 27), F925 (position 906 to 925), R529 (position 529 to 512), and R1392 (position 1406 to 1392) were used to obtain the sequences of both ends from both strands. About 200 bp from each individual primer was compared to the database containing the reference 16S rRNA gene sequences using the FASTA program (Genetics Computer Group Sequence Analysis Software Package; University of Wisconsin, Madison). If the sequences of the two ends showed the highest similarity to different reference strains but the sequences of both strands at the same end showed the highest similarity to the same reference strain, this clone was considered a chimera.
Primers F270 (position 246 to 261), F519 (position 512 to 529), F1099 (position 1099 to 1114), R350 (position 342 to 357), R925 (position 925 to 906), and R1540 (position 1541 to 1525) were used to obtain full 16S rRNA gene sequences. The sequences were assembled with PhredPhrap and Consed (University of Washington, Seattle) and compared to reference sequences using MAP, MAPSORT, and GAP (Genetics Computer Group). The error rate was the percentage of total examined clones that have a misincorporated nucleotide at the HhaI sites.Detection and elimination of heteroduplexes. To detect the heteroduplexes, an 8-µl aliquot of the PCR product from the clone with altered RFLP pattern was mixed with 2 µl of 50 mM EDTA (final concentration, 10 mM), denatured at 95°C for 5 min, and renatured at 25°C for 40 min (9, 35). The sample was separated on a 5% nondenatured polyacrylamide (49:1 ratio of acrylamide to bis) gel (16 by 20 cm) with a D Gene System (Bio-Rad) in 1× TBE buffer at 250 V at least for 3 h. The gel was stained with 0.5 µg of ethidium bromide per ml for 15 min, destained in 1× TBE for 30 min, and visualized by UV excitation. A heteroduplex was determined by comparing its banding pattern to those of reference homoduplex molecules. The clones showing extra bands that migrated more slowly than the homoduplex molecules but faster than single-stranded DNA molecules were considered heteroduplexes.
To remove heteroduplexes prior to cloning, 45 µl of amplified 16S rDNAs (Z-Taq) was mixed with 5 µl of 10× loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 25% Ficoll [type 400]), and separated on 5% nondenatured polyacrylamide gel. The bands corresponding to homoduplexes of 16S rDNAs were excised. The 16S rDNAs were recovered using a QIAEX II gel extraction kit (QIAGEN Inc., Valencia, Calif.) according to the manufacturer's instructions, except that the crushed gel strips were diffused twice with a 1.5× volume of diffuse buffer (0.5 M ammonium acetate 10 mM manganese acetate, 1 mM EDTA, and 0.1% sodium dodecyl sulfate) at 50°C for 30 min. 16S rDNAs were eluted in 40 µl of TE (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA) and concentrated to 20 µl by ethanol precipitation. The final concentration of the recovered 16S rDNAs was estimated on the 0.8% agarose gel, and about 20 ng was used for ligation. To digest heteroduplexes, 60 µl of amplified 16S rDNAs (LA-Taq) was concentrated to 6 µl by ethanol precipitation, and incubated with 1 µl (30 U) of T7 endonuclease I in a volume of 20 µl in a buffer containing 50 mM Tris (pH 8.0), 50 mM potassium glutamate, 10 mM MgCl2, 5 mM dithiothreitol, and 5% glycerol at 37°C for 20 min and then purified with the QIAEX II gel extraction kit and eluted in 20 µl of TE buffer (10 mM Tris [pH 8.0], 0.1 mM EDTA). Addition of 3'-A overhangs postamplification was carried out in a 20-µl reaction mixture containing 1× PCR buffer, 1.5 mM MgCl2, 0.1 mM dATP, and 0.5 U of Taq DNA polymerase at 65°C for 20 min. The resulting 16S rDNAs (about 20 ng) were directly used for ligation.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Detection of heteroduplexes by PAGE.
To test whether
heteroduplex molecules of nearly entire 16S rRNA genes (1.5 kb) can be
detected by polyacrylamide gel electrophoresis (PAGE) cloned 16S genes
from the two strains were mixed equally and subjected to PCR
amplification. The migration of heteroduplexes was retarded due to the
"bubble" formation between mismatches (Fig.
1). The decrease of the heteroduplex
mobility was inversely proportional to the sequence similarity of the
two parental molecules (Fig. 1A, lanes 6 to 11). These results
suggested that heteroduplexes, even those formed between distantly
related 16S rRNA genes (76% similar), could be detected by PAGE. Some
heteroduplexes of 16S rDNA formed between clones with misincorporated
nucleotides and their parental strains were also detectable on the 5%
nondenatured polyacrylamide gel (Fig. 1B, lanes 6 to 9).
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Effects of DNA polymerases on formation of PCR artifacts.
The
overall PCR-generated artifacts were enzyme dependent and significantly
different among the three Taq DNA polymerases examined. The
proportion of the overall PCR artifacts of LA-Taq was lower
than that of Z-Taq but significantly higher than that of
AmpliTaq (Table 2). No
significant difference in chimera was observed between Z-Taq
(8.7% ± 2.2%) and LA-Taq (6.2% ± 2.2%). However, the
frequency of chimeras for AmpliTaq was significantly lower
than those for Z-Taq and LA-Taq (Table 2).
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Effect of elongation time on formation of PCR artifacts. The total PCR artifacts decreased as the elongation time increased (Table 2). The highest percentage of PCR artifacts (25.5% ± 3.9%) was observed at an elongation time of 20 s. When the extension time increased to 4 min, the total PCR artifacts decreased to 16.1% ± 3.2%. Among the three types of PCR artifacts, the percentage of chimeras decreased dramatically from 10.3 to 3.7 as the elongation time increased. While the PCR-induced mutations varied from 9.6 to 12.8%, they were not significantly different among the three treatments. In addition, no significant difference in the percentage of heteroduplexes was observed when elongation time changed. About 2 to 3% heteroduplexes was observed for all three treatments (Table 2).
Effect of template concentration on formation of PCR artifacts. Since DNA template concentration will affect PCR amplification kinetics, it is expected that template concentration will have considerable effect on the formation of PCR artifacts. We found that the percentage of total PCR artifacts decreased from 17.3 to 10.5 as the template concentrations of each strain decreased from 10 ng to 100 pg (Table 2). This decrease was mainly due to the decrease in the error rates (from 8.0 to 2.5%). No significant difference in the percentages of heteroduplexes or chimeras was observed (Table 2).
Effect of PCR cycle number on formation of PCR artifacts. As expected, a positive correlation was observed between the formation of PCR artifacts and the number of PCR cycles, with PCR artifacts increasing from 13.0 to 20.8% as the cycles increased from 22 to 28. While the percentage of chimeras increased significantly as the cycle number increased, there was no significant change in the frequency of heteroduplexes. A slight increase in mutations was observed (Table 2).
Since AmpliTaq from Perkin-Elmer gave the lowest proportion of PCR artifacts, the effect of PCR cycle number on the formation of PCR artifacts was also examined with this enzyme. No difference of PCR artifacts was observed when the number of PCR cycles was 20 or less, while the total PCR artifacts increased dramatically as the cycle number increased above 20. No chimera was detected when the cycle number was 15, whereas 4.3% chimeras was observed for 30 PCR cycles. Slight increases were observed in both mutations (1.9 to 3.7%) and heteroduplexes (0.6 to 2.5%). Similar to that described above, the overall PCR artifacts were significantly lower in the amplifications with AmpliTaq than those of Z-Taq (Table 2).Effects of species diversity on formation of PCR artifacts.
Total PCR artifacts increased as the species diversity increased (Table
3). Among 162 clones examined, four PCR
artifacts (2.5%) were detected in the four-species community. All
these four clones were mutations of P39. Six artifacts (3.7%) were
found in the seven-species community; three were mutations, and the other three were heteroduplexes. There were nine PCR artifacts (5.6%)
in the 10-species community. Three were mutations, and six were
heteroduplexes. No chimeric molecule was detected in all three model
communities.
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Elimination of heteroduplexes by polyacrylamide gel purification or
T7 endonuclease I digestion.
Since heteroduplex molecules migrate
more slowly than homoduplex molecules in polyacrylamide gel, the
heteroduplex molecules should be separated from the PCR products by
PAGE. To test the effectiveness of this purification method, a clone
library was constructed using the 16S rDNAs purified from the
polyacrylamide gel. No heteroduplexes were observed in this
library, whereas 3.7% was detected in the control library (Table
4).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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PCR-based cloning approaches are powerful tools for analyzing microbial community diversity despite intrinsic problems of bias and artifacts. With careful planning and experimental condition control, the artifacts can be minimized. Although the frequencies of artifacts observed in this study cannot be extrapolated to other studies due to differences in experimental conditions, they do provide valuable information for improving the methodologies of PCR-based cloning studies.
The effects of three types of PCR artifacts on the 16S-based cloning studies depend on experimental purpose. Single-base mutations may have little or no effect on the overall tree topology when the entire 16S rRNA gene sequences are compared. Clean sequences may not be obtained if the clone resulted from a heteroduplex; thus, it should not be a concern when the sequence is required for the analysis. However, all three types of PCR artifacts can have serious impacts when the RFLP or terminal RFLP analysis DGGE, or TGGE, etc., is used for microbial community analysis.
The existence of heteroduplexes in a cloned 16S rRNA gene library has been an unappreciated problem and can lead to overestimating the diversity of a microbial community. We found that the occurrence of heteroduplexes indirectly correlated with DNA polymerases. More heteroduplexes were observed in the amplifications with Z-Taq than with AmpliTaq. This could be due to the fact that more PCR products were synthesized by Z-Taq than by AmpliTaq. High PCR product production will favor heteroduplex formation. This explanation is further supported by the observed increase in proportion of heteroduplexes as the cycle number and template concentrations increased. Heteroduplex frequency also appeared to be a function of species diversity. The frequency of heteroduplexes in the 10-species community was about two to four times higher than those of 7- or 4-species communities. This could be due to the fact that the probability of annealing between the two strands from the same origin decreases as the number of the heterogeneous genes increases. The corresponding increase in heteroduplexes with species diversity is potentially a problem for analyzing natural microbial communities, which may have hundreds to thousands of phylotypes. Besides, there is usually more than one rrn copy in a genome. Theoretically, the frequency of forming heteroduplex molecules between different copies of the 16S rRNA gene in the same genome is higher than that between different 16S rRNA genes, because a heteroduplex should be more stable when the two parental genes have higher sequence similarity.
We proved that heteroduplex molecules of the entire 16S rRNA gene (1.5 kb) could be effectively detected by PAGE in a wide similarity range of 16S rRNA genes. However, the conditions for detecting such long fragments are different from those for DGGE, single-strand conformation polymorphism analysis, or heteroduplex mobility assay. We found that a gel with a lower cross-linking ratio (49:1), which yields a bigger pore size, should be used. Including 10% glycerol in the polyacrylamide gel helped to detect heteroduplexes, but a low concentration of urea in the gel did not. In addition, the conditions for forming heteroduplexes by denaturation-renaturation were also critical. We found that quickly cooling to 25°C for renaturation was much better than slowly cooling to 25°C (37) or quickly cooling on ice (9). Renaturation on ice resulted in more single-stranded DNA fragments. Including 10 mM EDTA in denaturation-renaturation buffer was helpful. However, we had difficulty forming heteroduplexes by denaturation at 98°C for 7 min and renaturation at 60°C for 40 min (12). We could detect the heteroduplexes formed between both closely (98.5% similar) and distantly (76% similar) related 16S rRNA genes by 5% nondenatured polyacrylamide gel.
We recommend the elimination heteroduplexes prior to cloning. It is possible to use an enzyme such as T7 endonuclease I to cut the bubble in a heteroduplex and further destroy it (22). However, the experimental conditions for this treatment are critical. Low concentrations of enzyme or short incubation time do not remove all heteroduplexes, whereas high concentrations of enzyme or long incubation time can digest the homoduplex molecules. Also, postamplification with Taq is required for this approach to generate an A overhang for TA cloning. PAGE was also effective in removing heteroduplex molecules; however, this approach may be difficult for separating the heteroduplexes formed between highly related strains or heteroduplexes having very close conformations to the parental homoduplex molecules.
PCR-generated mutation is another little-recognized problem for 16S
rRNA gene-based cloning studies. In general, misincorporated nucleotides in the PCR products is not a big concern since the errors
are distributed randomly over the amplified fragment. Theoretically, less than one misincorporated nucleotide is expected when the entire
16S rRNA gene is amplified using an enzyme with an average of fidelity
such as 8 × 10
6 /base/replication. However, the
error rate observed in this study was much higher than predicted. The
highest error rate was observed for Z-Taq; however, the
fidelity of Z-Taq (8.6 × 106
/base/duplication) is very close to that of AmpliTaq. These
results indicated that the PCR-generated errors were not merely the
consequences of infidelity of Taq polymerases. Since both
Z-Taq and LA-Taq have higher processivity than
AmpliTaq, we suspected that the higher error rate might be
caused by a lack of PCR reagents, especially dNTPs. This explanation is
supported by two observations: first, more PCR products were
synthesized by Z-Taq and LA-Taq compared to
AmpliTaq when equal units of enzymes were used; second, more PCR-induced mutations were observed when more templates were used (Table 2).
PCR amplification fidelity is affected by many factors
not only the
enzyme used but also buffer conditions, divalent metal cations, and
thermal cycling parameters. It was reported that Taq
fidelity decreased when the concentration of Mg2+ was in
great excess compared to total dNTPs (11). The
Mg2+ and dNTP concentrations used in this study were the
optimum concentrations recommended by the manufacturers. The rates of
excess of Mg2+ over total dNTPs with Z-Taq,
LA-Taq, and AmpliTaq were 2.2, 0.9, and 0.7 mM,
respectively. All these assays were within the range of high-fidelity
conditions described by Eckert et al. (11). Whether it has
true impact on the high error rate needs to be further examined.
We also observed that certain types of artificial RFLP patterns that
were caused by a misincorporated single nucleotide appeared in many
independent amplifications with each of the three Taq DNA
polymerases. To better understand this phenomenon, eight clones, each
with a distinct artificial RFLP pattern that most frequently arose,
were studied in detail. Clones 16, 6, 34, and 7 gained an
HhaI site whereas clones 15, 40, 14, and 35 lost an
HhaI site, all due to a base substitution (Table
5). Moreover, both error sites of clone
15 (mutation of C1-4) and clone 14 (mutation of B9-12) were at the
E. coli position 1109, where C was in a loop (Table 5). The
16S rDNA sequences of the two clones were significantly different
(79.4% similar). Hence, we suspect that the secondary structure of 16S
rRNA gene contributed to the high error rate observed.
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Consistent with the results of Wang and Wang (34, 35), we found that the longer extension times and fewer PCR cycles decreased the frequency of chimeras. The percentages of chimeras found in this study, however, were more than three times lower than those observed by Wang and Wang (34, 35), probably due to different experimental systems and conditions. For example, they used pairs of cloned 16S rRNA genes with sequence similarity of 99.3, 86, and 82%, whereas sequence similarity varied from 76 to 89% in our four-species model community. Also, our chimera detection method could be less sensitive since we detected only an altered RFLP pattern. In theory, using Taq DNA polymerase with higher processitivity should lower the frequency of chimeras, since chimeric molecules are mainly caused by incomplete synthesis during the PCR cycle (24, 38). Conversely, the highest frequency of chimeras was observed for Z-Taq, which has the highest processitivity. Likely, other factors contributed to the formation of chimeras. For example, an undenatured region or secondary structure in the templates will make it difficult for DNA polymerase to read through, causing termination of DNA synthesis.
While some tools are available for detecting chimeras and heteroduplexes after cloning, no tools are available for identifying PCR-generated single-base mutations in natural samples. Thus, it is critical to minimize PCR artifact formation prior to cloning. PCR cycling is one key parameter to reducing all three types of PCR artifacts. We suggest that the PCR amplification for any cloning-based community studies be performed with as few cycles as possible (http://www.esd.ornl.gov/people/zhou/zhou.html). The appropriate cycle number will depend on the amount of template used, amplification efficiency, and existence and degree of inhibitory substances and thus should be determined experimentally. To minimize PCR artifacts, we suggest using the PCR products prior to or during the exponential period for cloning. To obtain enough products for cloning, we suggest combining multiple amplifications followed by concentration with ethanol precipitation. Mixing PCR products from independent amplifications can also help to minimize experimental errors and amplification bias. The concentrated sample can then be quantified and used for constructing 16S rRNA gene library. Because the extent of 16S rRNA gene artifacts can never be known in a natural sample, we suggest that interpretations be focused on comparative studies with replication and under identical PCR conditions.
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ACKNOWLEDGMENTS |
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We thank Joe-Chang Cho for discussion and advice on heteroduplex detection.
This research was supported by the Natural and Accelerated Bioremediation Research and the Biotechnology Investigations-Ocean Margins Program, Office of Biological and Environmental Research, The United States Department of Energy. Oak Ridge National Laboratory is managed by University of Tennessee-Battelle LLC for the Department of Energy under contract DE-AC05-00OR22725, and the Center for Microbial Ecology is funded by the National Science Foundation, DEB-9120006.
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FOOTNOTES |
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* Corresponding author. Mailing address: Environmental Sciences Division, Oak Ridge National Laboratory, P. O. Box 2008, Oak Ridge, TN 37831-6038. Phone: (865) 576-7544. Fax: (865) 576-8646. E-mail: zhouj{at}ornl.gov.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, February 2001, p. 880-887, Vol. 67, No. 2
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.2.880-887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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