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Applied and Environmental Microbiology, February 2000, p. 549-557, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Peptide Nucleic Acid-Mediated PCR Clamping as a Useful Supplement
in the Determination of Microbial Diversity
Friedrich
von
Wintzingerode,1
Olfert
Landt,2
Angelika
Ehrlich,3 and
Ulf
B.
Göbel1,*
Institut für Mikrobiologie und Hygiene,
Universitätsklinikum Charité, 10117 Berlin,1 TIB MOLBIOL Syntheselabor,
10829 Berlin,2 and Forschungsinstitut
für Molekulare Pharmakologie im Forschungsverbund Berlin
e.V., 10315 Berlin,3 Germany
Received 9 July 1999/Accepted 10 November 1999
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ABSTRACT |
Peptide nucleic acid (PNA)-mediated PCR clamping (H. Ørum, P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, and C. Stanley, Nucleic Acids Res. 21:5332-5336, 1993) was introduced as a novel procedure to selectively amplify ribosomal DNAs
(rDNAs) which are not frequently found in clone libraries generated
by standard PCR from complex microbial consortia. Three different
PNA molecules were used; two of these molecules (PNA-ALF and
PNA-EUB353) overlapped with one of the amplification primers, whereas
PNA-1114F hybridized to the middle of the amplified region. Thus, PCR
clamping was achieved either by competitive binding between
the PNA molecules and the forward or reverse primers (competitive
clamping) or by hindering polymerase readthrough
(elongation arrest). Gene libraries generated from mixed rDNA
templates by using PCR clamping are enriched for clones that do not
contain sequences homologous to the appropriate PNA oligomer. This
effect of PCR clamping was exploited in the following two ways: (i)
analysis of gene libraries generated by PCR clamping with PNA-ALF
together with standard libraries reduced the number of clones which had
to be analyzed to detect all of the different sequences present in an
artificial rDNA mixture; and (ii) PCR clamping with PNA-EUB353 and
PNA-1114F was used to selectively recover rDNA sequences which
represented recently described phylogenetic groups (NKB19, TM6, cluster
related to green nonsulfur bacteria) from an anaerobic, dechlorinating consortium described previously. We concluded that PCR clamping might
be a useful supplement to standard PCR amplification in rDNA-based
studies of microbial diversity and could be used to selectively
recover members of undescribed phylogenetic clusters from complex
microbial communities.
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INTRODUCTION |
Culture-independent analysis of
PCR-amplified 16S rRNA gene (rDNA) libraries is a powerful approach
for determining the diversity of complex microbial environments
(7, 8). By using PCR-primers that target conserved regions
of the 16S rDNA sequences can be retrieved both from well-known
bacterial or archaeal phyla and from phylogenetic groups represented
exclusively by uncultured microorganisms (10).
Here we describe peptide nucleic acid (PNA)-mediated PCR
clamping (18) as a novel approach for generating rDNA
clone libraries from environmental samples. PNA-mediated PCR clamping
relies on the following two unique properties of PNA oligomers: (i)
PNA-DNA duplexes generally have greater thermal stability than the
corresponding DNA-DNA duplexes (17); and (ii) PNA oligomers
are not recognized by DNA polymerases and consequently cannot serve as
primers during PCR amplification (18). Bound PNA does not
inhibit PCR completely but reduces amplification efficiency. PCR
clamping of mixed DNA templates (e.g., total rDNA of a
microbial community) inhibits amplification of sequences which are
perfectly homologous to the respective PNA oligomer, which results in
preferential amplification of sequences with mismatches to the PNA.
Thus, PCR clamping introduces a preferential bias to selectively enrich
nontarget sequences of a mixed template. By using both an artificial
rDNA mixture and natural community rDNA we found that this
ability of PCR clamping can supplement standard PCR amplification in
rDNA-based studies of microbial diversity.
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MATERIALS AND METHODS |
Bacterial strains and environmental rDNA templates.
The
bacterial strains and cloned rDNAs from an environmental source
used as references in PCR clamping experiments are listed in Table
1. Reamplified rDNA inserts were
generated from a 16S rDNA library as described previously
(23). Full-length 16S rRNA genes of bacterial reference
strains were PCR amplified by using primers TPU1 and RTU8 and the
following hot-start protocol: initial denaturation at 98°C for
30 s and at 93°C for 2 min, addition of AmpliTaq polymerase
(Perkin-Elmer, Weiterstadt, Germany), 25 cycles consisting of
denaturation at 93°C for 1 min, primer annealing at 53°C for 1 min,
and elongation at 72°C for 2 min, and a final elongation step
consisting of 72°C for 7 min. Amplification products were cloned as
described below and were reamplified by using vector-specific primers
M13(
40)F and M13(24)R. For reamplification we used the conditions
described above except that the hot start was omitted and the initial
denaturation step consisted of denaturation at 95°C for 5 min. The
resulting PCR amplicons were purified with a silica suspension
(5) and Geneclean spin filters (Dianova, Hamburg, Germany).
For PCR clamping experiments performed with PNA-EUB353 and PNA-1114F
amplified community 16S rDNA was used as the template. The DNA
concentrations in all of the rDNA controls were determined
spectrophotometrically by using an Ultrospec III photometer (Pharmacia,
Freiburg, Germany).
Synthesis of PNA.
PNA were synthesized with a model 8900 Expedite nucleic acid synthesizer (PerSeptive Biosystems, Framingham,
Mass.) by using an amide resin, Fmoc-protected monomers (Perkin-Elmer),
and the protocol recommended by the manufacturer (PNA II method;
PerSeptive). The resin was washed with dichloromethane (Merck,
Darmstadt, Germany), and the products were deprotected with 80%
trifluoroacetic acid (TFA)-20% m-cresol (Merck) for 90 min
at room temperature (21°C). The product was precipitated with 6 volumes of ether (Merck), incubated for 10 min at 18°C, and
pelleted by centrifugation. The pellet was washed with ether, dried
with a Speed Vac apparatus, resuspended in 50 µl of 0.1% TFA,
resolved completely by adding 200 µl of acetic acid, and precipitated
again as described above. The dried pellet was resolved with 500 µl
of 0.1% TFA and was incubated for 1 h at 60°C. The PNA was
purified by reversed-phase high-performance liquid chromatography by
using a pepRPC HR5/5 column (Amersham-Pharmacia, Freiburg, Germany)
with aqueous 0.1% TFA and 0.08% TFA in acetonitrile (PROLIGO,
Hamburg, Germany). The fractions that were collected were lyophilized
and dissolved in 0.1% TFA. The yield was determined by measuring
absorbance at 260 nm. The purity of the PNA oligomers was confirmed by
mass spectroscopy (PerSeptive).
PNA-mediated PCR clamping.
The DNA and PNA oligomers used in
this study are shown in Table 2. All PCRs
were performed by using a total volume of 25 µl and a Trioblock
thermocycler (Biometra, Göttingen, Germany). Each reaction
mixture contained 1× PCR buffer (Gibco; Roche Diagnostics, Mannheim,
Germany), 7.5 pmol of primer, 1.5 mM MgCl2, each
deoxynucleoside triphosphate (Roche Diagnostics) at a
concentration of 25 µM, 9 pmol of PNA (omitted in the control PCR),
and 0.5 or 1.25 U of AmpliTaq polymerase (Perkin-Elmer). The template
concentrations used in most reaction mixtures were 16 pg/µl for
reference rDNA and 80 pg/µl for community rDNA; the only
exception was the experiment in which PCR clamping with PNA-ALF was
used, in which a mixture of different templates was used, as described
below. The cycling conditions used were similar in all of the PCR
clamping experiments (variations are indicated in
parentheses), as follows: initial denaturation at 95°C for 5 min,
followed by 20 cycles consisting of denaturation at 93°C for 1 min
(92°C for PNA-EUB353), PNA annealing at 70°C for 1 min, primer
annealing at 53°C for 1 min (61°C for PNA-EUB353), and elongation
at 72°C for 1 min (90 s for PNA-1114F) and a final
elongation step consisting of 72°C for 7 min (only for PNA-EUB353).
Amplification products were separated by agarose gel electrophoresis
and were visualized by staining the gels with ethidium bromide (100 µg/liter).
Analysis of amplicons generated by PCR clamping. (i) PCR
cloning.
PCR amplicons were directly ligated into vector
pCR2.1 of a TA cloning kit by following the instructions of the
manufacturer (Invitrogen, de Schelp, The Netherlands). Amplicons
obtained from PCR clamping with PNA-1114F were purified by
preparative gel electrophoresis by using a QiaQuick gel
extraction kit (Qiagen, Hilden, Germany). To compensate for the
loss of DNA during purification, excised agarose bands from three
PCR were combined and eluted in 50 µl of the buffer supplied.
(ii) Dot blot hybridization.
For dot blot
hybridization 16S rDNA inserts of randomly selected recombinant
Escherichia coli clones were PCR amplified by using M13
primers as described above. Alternatively, plasmid DNA was isolated by
using a GFX plasmid preparation kit (Amersham-Pharmacia, Freiburg,
Germany) as recommended by the manufacturer. The presence of inserts of
the expected size was determined by digesting plasmid DNA with
EcoRI. Plasmid DNA or PCR amplicons were heat denatured (95°C, 4 min) and immediately cooled on ice. Aliquots (1.5 µl) were
spotted onto a positively charged nylon membrane (Roche Diagnostics) and fixed by UV cross-linking. Prior to hybridization with FAM-labeled oligonucleotide probe EUB353, membranes were prehybridized with 25 ml
of standard hybridization buffer containing 5× SSC (1× SSC is 0.15 M
NaCl plus 0.015 M sodium citrate), 0.1% sodium lauroylsarcosine, 0.02% sodium dodecyl sulfate, and 1% blocking reagent (Roche
Diagnostics) at 42°C for 30 min. Hybridization was performed with 25 ml of standard hybridization buffer containing 60 pmol of the probe at
42°C for 2 h and this was followed by two stringency washes at
54°C with 40 ml of 6× SSC-0.1% sodium dodecyl sulfate for 15 min.
Digoxigenin (DIG)-labeled polynucleotide probes were generated and
hybridization was performed as described previously (23), except that two probes were used simultaneously. Hybrids were detected
on X-ray film by chemoluminescence by using the reagents of a DIG
luminescence detection kit (Roche Diagnostics). To detect the
FAM-labeled oligonucleotide, the anti-DIG conjugate was replaced by an
alkaline phosphatase coupled to anti-FAM immunoglobulin G antibodies
(TIB MOLBIOL, Berlin, Germany).
(iii) Sequencing and phylogenetic analyses.
Plasmid DNA or
silica-purified PCR products were used as templates for cycle
sequencing in which we used a Thermo Sequenase fluorescently labeled
primer cycle sequencing kit (Amersham-Pharmacia) and fluorescently
labeled universal M13 primers and 16S rDNA-specific primers.
Sequencing reactions were analyzed by using an automated model LICOR
DNA4000L sequencer (MWG-BIOTECH, Ebersberg, Germany). Phylogenetic
analyses were performed by using the ARB software package
(http://www.mikro.biologie.tu-muenchen.de/pub/ARB/documentation/arb.ps). A phylogenetic tree (see Fig. 5) was constructed by using the Jukes-Cantor correction (11) and the neighbor-joining method (20) with 1,000 bootstrap resamplings. Sequences were
analyzed for possible chimeric structures by the fractional treeing
method as described previously (23).
Nucleotide sequence accession numbers.
Nineteen nucleotide
sequences of rDNA clones and reference strains have been deposited
in the EMBL, GenBank, and DDBJ nucleotide sequence databases under
accession no. AJ387887 to AJ387905.
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RESULTS |
PCR clamping of an artificial rDNA mixture.
We designed a
competitive clamping experiment in which primer TPU1 and the PNA-ALF
oligomer, which had an eight-base overlap, were used (Table 2). First,
the specificity was tested by using different reference rDNA
templates. As shown in Fig. 1, PCR
amplification of completely complementary rDNAs (clones SJA-9 and
SJA-105) was suppressed by PNA-ALF, whereas rDNA of clone SJA-53 or
Verrucomicrobium spinosum (one mismatch each) and rDNA
of clone SJA-186 (two mismatches) were amplified.
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FIG. 1.
Agarose gel electrophoresis analysis of
products of various PCR clamping reactions. Lanes 1 through 5, PCR
performed with PNA-ALF and reference rDNAs (V. spinosum [one mismatch], SJA-186 [two mismatches], SJA-53
[one mismatch], SJA-9 [no mismatch], SJA-105 [no mismatch]);
lanes 6 through 8, PCR performed with PNA-EUB353 and reference
rDNAs (V. spinosum [two mismatches], strain OLB-1 [no
mismatch], C. gallinarum [no mismatch]); lanes 9 through
16, duplicate PCR performed with (+) or without () PNA-1114F
and reference rDNAs (V. spinosum [no mismatch], strain
OLB-1 [one mismatch], SJA-131 [two mismatches], SJA-4 [three
mismatches]); lanes M, length standard (100 bp plus; MBI-Fermentas,
St.-Leon Roth, Germany). The sizes of relevant bands are shown on the
left and right.
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Second, an artificial rDNA mixture was analyzed. The sample used
contained a 100-fold excess of target rDNAs (SJA-9 and SJA-105)
compared with nontarget rDNAs (SJA-186 and SJA-53). As controls,
PCR without PNA-ALF were performed in parallel. All PCR and cloning
reactions were performed in triplicate, which resulted in three
independent clone libraries each for both clamped PCR (clone libraries
A1 to A3) and control PCR (clone libraries A4 to A6) (Fig.
2).
A total of 68 randomly selected
clones (34 clones from each set)
were analyzed. As shown in Fig.
2,
simultaneous dot blot hybridization
performed with DIG-labeled
polynucleotide probes specific for
SJA-186 and SJA-53 revealed that 28 of the 34 clones (82.4%) in
the libraries derived from PCR clamping
were positive, while only
1 positive clone (2.9%) was detected among
the 34 clones derived
from standard PCR.
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FIG. 2.
Analysis of an artificial rDNA mixture by
competitive clamping: schematic diagram of PCR clamping performed with
PNA-ALF and results of dot blot hybridizations with polynucleotide
probes specific for SJA-53 and SJA-186. Positions P1 to K3, selected
clones from libraries A1 to A3 (PCR clamping); positions L3 to G5,
selected clones from libraries A4 to A6 (control PCR). The following
16S rDNAs were used as controls: SJA-9 (position I5), SJA-53
(position J5), SJA-105 (position K5), and SJA-186 (position M5).
Asterisks indicate positions where no samples were applied.
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PCR clamping of total community rDNA.
To demonstrate the
ability of PCR clamping to selectively recover rDNA sequences of
recently described phylogenetic groups from total community rDNA,
we used the rDNA amplicon of an anaerobic dechlorinating consortium
as the template; it had been shown previously that this consortium
included several representatives of recently described phylogenetic
groups (23). The following two strategies were used;
(i) competitive clamping with primers TPU1 and 365R and
PNA-EUB353, which had a binding site that overlapped with the
binding site of the reverse primer 365R; and (ii) elongation arrest
performed with primers TPU1 and RTU8 and PNA-1114F, which bound to the
middle of the PCR target region (Fig.
3B). The specificities of the two
PCR clamping approaches were tested with individual reference
rDNA templates before complex target rDNAs were used.
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FIG. 3.
Schematic diagram of PNA-mediated PCR clamping of 16S
rDNA amplification. (A) Competitive clamping: inhibition of PCR
amplification by PNA-ALF-mediated exclusion of primer TPU1. (B)
Elongation arrest: inhibition of PCR amplification by binding of
PNA-1114F to an internal target sequence, which prevents readthrough by
the Taq polymerase. In both cases amplification proceeds
only if one or more base substitutions in the binding sites of
appropriate PNA molecules are present.
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While PCR amplification of complementary sequences of strain OLB-1 and
Carnobacterium gallinarum was specifically inhibited
by PNA-EUB353, template rDNA derived from
V. spinosum (two mismatches)
was readily amplified (Fig.
1).
Compared to the competitive PCR
clamping approach, which resulted in
complete suppression of PCR
amplification, PNA-1114F-mediated
elongation arrest led to reduced
amplification of target rDNAs
but not to complete amplification
arrest (Fig.
1).
The efficiency of competitive PCR clamping by PNA-EUB353
when total community rDNA was used was analyzed as shown in Fig.
4. A standard PCR (without a PNA
oligomer) served as the control.
All PCR and cloning reactions were
performed in triplicate, which
resulted in clone libraries B1 to
B3 (PCR clamping) and B4 to
B6 (control PCR). A total of 46 clones in libraries B1 to B3 and
45 clones in libraries B4 to B6 were
randomly chosen and used
for further analysis. Dot blot
hybridization with FAM-labeled
oligonucleotide probe EUB353
revealed no positive clones among
the 46 clones in libraries derived
from PCR clamping, whereas
20 (44.4%) of the 45 clones obtained from
the control PCR were
hybridization positive (Fig.
4). To verify the
hybridization results
and to perform an additional phylogenetic
analysis, 17 EUB353-negative
clones in libraries B1 to B3 and 9 positive clones were sequenced.
Sequencing of the oligonucleotide
EUB353 target site confirmed
the specificity of the dot blot
hybridization procedure and revealed
that all of the EUB353-negative
clones had several base substitutions
(Table
3). A subsequent phylogenetic analysis
showed that 16
of 17 EUB353-negative clones in libraries B1 to B3
either were
affiliated with a phylogenetic cluster close to the green
nonsulfur
(GNS) bacteria (
10) or were related to rDNA
sequence NKB19 (
14),
which was not closely related to any of
the phylogenetic groups
described previously (Table
4). In contrast, seven of nine
EUB353-positive
clones either were representatives of previously
described proteobacterial
SJA clone families or were identical to the
16S rDNA sequence
of
Pseudomonas sp. strain RNA-111,
which had been isolated from
the same community previously
(
24).
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FIG. 4.
Analysis of community rDNA by competitive clamping:
schematic diagram of PCR clamping performed with PNA-EUB353 and results
of dot blot hybridizations with oligonucleotide probe EUB353. Positions
A1 to H4, selected clones from libraries B1 to B3 (PCR clamping);
positions I4 to A8, selected clones from libraries B4 to B6 (standard
PCR). The following 16S rDNAs were used as controls: SJA-22
(position B8), SJA-43 (position C8), SJA-131 (position D8),
Pseudomonas aeruginosa ATCC 25330 (position G8), C. gallinarum DSM 4847T (position H8), and V. spinosum DSM 4136T (position I8). Asterisks indicate
positions where no samples were applied.
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TABLE 3.
Competitive clamping of total community rDNA:
analysis of PNA-EUB353 nontarget sequences (E. coli
positions 336 to 355) of clones belonging to gene libraries B1 to B3
generated by PCR clamping and of selected reference sequences
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TABLE 4.
Phylogenetic affiliations of 17 EUB353-negative clones
and 9 EUB353-positive clones selected from 16S rDNA clone libraries
generated by competitive clamping (clone libraries B1 to B3) and
standard PCR of total community rDNA, respectively
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PCR clamping performed by the elongation arrest strategy with PNA-1114F
and primers TPU1 and RTU8 was done in triplicate and
resulted in three
clone libraries, which were designated C1 to
C3. The effect of PCR
clamping was measured by using clone library
SJA as the control; this
clone library had been generated previously
by standard PCR from total
community DNA by using the same broad-spectrum
primers (
23).
Twenty-four randomly chosen clones in libraries
C1 to C3 were sequenced
(~800 to 1,500 bp) in order to determine
both their phylogenetic
affiliation and the sequence of the PNA-1114F
target site. This
analysis revealed that 11 of the 24 clones (45.8%)
were
PNA-1114F negative and had up to three base substitutions
in the
PNA-1114F target site (Table
5). Thus,
when PNA-1114F-mediated
elongation arrest was used, an
approximately sixfold relative
increase (6.9%
PNA-1114F-negative clones among SJA clones) in
nontarget sequences was
observed compared to the SJA clone library
generated by standard PCR
alone. Sequences of nontarget clones
either were nearly identical
(>99%) to the sequence of clone SJA-4
in the TM6 cluster
(
19) or were affiliated with the phylogenetic
cluster
related to GNS bacteria (Fig.
5). One
clone was identified
as a sequence chimera.
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TABLE 5.
PCR clamping by elongation arrest: analysis of PNA-1114F
target sequences (E. coli positions 1097 to 1116) of
selected clones belonging to gene libraries generated by PCR clamping
and reference sequences
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FIG. 5.
Phylogenetic dendrogram showing clusters mentioned in
this study. For sequences marked with an asterisk a corresponding
rDNA clone (level of sequence similarity, >99%) was recovered by
competitive clamping with PNA-EUB353 (Table 5). For sequences marked
with a less-than sign the corresponding rDNA clone was obtained by
elongation arrest with PNA-1114F. PNA-1114F nontarget clones C1-28 and
C2-12 could not be affiliated with any of the previously described SJA
clone families in the cluster related to GNS bacteria and were included
in the dendrogram. 16S rDNA clone sequences SJP-2 and SJP-3
(~1,300 bp) were amplified from total community rDNA by a
seminested PCR performed with forward primer NKB19F (5'
GCTGCAAGGCGTCGCCG 3') derived from NKB19-related clones B1-9,
B1-11, and B3-6 and universal reverse primer RTU8. Branch points
supported by bootstrap values of >74% are indicated by solid circles.
Open circles indicate branch points supported by bootstrap values
between 50 and 74%. Branch points without circles were not resolved
(bootstrap values, <50%). Scale bar = 10% sequence
divergence.
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DISCUSSION |
Culture-independent analysis of PCR-amplified 16S rRNA gene
libraries for studying the diversity of complex microbiota has become
an important tool in microbial ecology. This method includes analysis
of both cultured and uncultured microorganisms (7, 8).
Various investigations of environmental and man-made microbial habitats
not only have increased our knowledge concerning the phylogenetic
diversity of well-known bacterial or archaeal phyla but also have
revealed the presence of novel phylogenetic groups represented
exclusively by uncultured microorganisms (for a review, see reference
10). However, the laborious and time-consuming steps
involved in analysis of gene libraries present a serious problem since
usually a large number of clones have to be screened to detect not only
the dominant sequences but also the less abundant clones in a rDNA
gene library. Here, we used PNA-mediated PCR clamping, which has been
used to suppress certain variants in order to identify clinical
relevant polymorphisms (3, 16, 18, 21), to reduce
amplification of abundant sequences from a standard clone library and
to enrich rare sequences, which were less homologous to the PNA
oligomer used. PNA-mediated PCR clamping might be a useful supplement
to standard PCR when it is included in the following strategy: (i)
generation of a rDNA clone library by standard PCR and
determination of the abundant sequence types; (ii) design of a PNA
oligomer(s) that targets the abundant sequences; and (iii) analysis of
an additional rDNA clone library generated by PNA-mediated PCR
clamping. In this study we used PCR clamping for both possible
strategies, competitive clamping that attacked either the forward
primer (PNA-ALF) or the reverse primer (PNA-EUB353) and the elongation
arrest approach (PNA-1114F).
PCR clamping of an artificial rDNA mixture.
PCR clamping
to selectively enrich nontarget sequences was first used with an
artificial mixture containing 1% SJA-53 rDNA and 1% SJA-186
rDNA. Only one of these rDNAs was detected in 34 clones
belonging to gene libraries A4 to A6 generated by standard PCR. This
changed, however, if PNA-ALF, which specifically binds to SJA-9 and
SJA-105 rDNAs, was added to the PCR mixture. In this case, the
initially less numerous SJA-53 and SJA-186 rDNAs clearly dominated,
resulting in clone libraries A1 to A3 (28 of 34 clones). This example
demonstrated the potential of PCR clamping as a supplement for standard
PCR in rDNA-based diversity studies. When both PCR approaches were
used, analysis of a few clones was sufficient to detect all of the
different sequences in the artificial mixture, and consequently
screening a large number of standard PCR clones was not necessary.
PCR clamping of natural community rDNA.
In a recent
phylogenetic analysis of an anaerobic, trichlorobenzene-dechlorinating
microbial consortium (23), it was shown that several
rDNA clones (SJA sequences) were affiliated with recently described
phylogenetic clusters (clusters OP10 [9], WS1
[6], and TM6 [19], and a cluster
related to the GNS bacteria [10]) and were closely
related to rDNA sequences of uncultivated bacteria in another
anaerobic, dechlorinating community (6). In most of
these SJA sequences there were variations in highly conserved
rDNA signature sites for the domain Bacteria
(EUB353 site [1] and 1114F site
[13]), and consequently the sequences were
designated EUB353/1114F-negative sequences. Future studies of
anaerobic, dechlorinating microbial consortia should focus on
EUB353/1114F-negative sequences since they might represent indicator
organisms for the dechlorination process (23). However, for
such studies analysis of standard rDNA clone libraries alone is not
reasonable since EUB353/1114F-negative sequences accounted for only
about 19% of all of the SJA sequences analyzed; consequently, screening a large number of clones will be necessary to detect these sequences. Therefore, PCR clamping with PNA-EUB353 and PNA-1114F was tested to determine its ability to selectively recover
EUB353/1114F-negative sequences from previously studied total community rDNA.
Indeed, PCR clamping with PNA-EUB353 and bacterium-specific primers
specifically amplified rDNA sequences belonging to the
cluster
related to the GNS bacteria and sequences affiliated with
rDNA
sequence NKB19, which had not been detected among the SJA
clones
previously (
23).
Compared to competitive clamping with PNA-EUB353, clamping
by elongation arrest with PNA-1114F was less effective since it
resulted in less enrichment of rDNA sequences affiliated with
the
GNS bacterium-related cluster and with cluster TM6. This might
have
been due to interactions among the three reaction molecules
(PNA, DNA,
and
Taq polymerase), which were obviously more complex
than
the interactions between PNA and DNA in competitive clamping.
For
example, our efforts to use PNA-EUB353 (16-mer) or an extended
PNA
(19-mer) for PCR clamping by elongation arrest consistently
failed. This finding is consistent with previously described problems
with this PCR clamping type (
18; O. Landt,
unpublished data).
However, compared to competitive clamping, clamping
by elongation
arrest offers greater flexibility in chosing target sites
for
PNA oligomers and primers. Our results for both sensitivity and
specificity of PCR clamping confirm the previous findings of Behn
and
Schuermann (
2) that PCR clamping selectively amplifies
nontarget sequences in the presence of a high background level
of
target sequences. In the study of these authors PCR clamping
followed
by SSCP analysis allowed detection of
p53 gene mutations
even in samples with a 200-fold excess of wild-type genes. Similar
to
our data, most PCR clamping protocols result in specific binding
of PNA
oligomers at 68 or 70°C (
2,
3,
12,
16,
21).
The fact that
binding of PNA oligomers is less sensitive to base
composition and
length of the oligomer definitely simplifies the
design of PCR clamping
experiments. We are aware that biases introduced
by standard PCR
amplification and subsequent cloning (for a review,
see reference
22) are not compensated for with this method.
Furthermore, less abundant sequences that are fully complementary
to the PNA oligomer are not selectively amplified by PCR clamping.
However, as shown in this study, PCR clamping might be a useful
supplement for standard PCR. Further work with different PNA
oligomers
and other complex microbiota is urgently needed to fully
explore
the potential of PNA-mediated PCR clamping in
rDNA-based studies
of microbial
diversity.
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ACKNOWLEDGMENTS |
We thank Heidemarie Hans (Forschungsinstitut für Molekulare
Pharmakologie, Berlin im Forschungsverbund Berlin e.V.) for excellent technical assistance and Claudia Bergmüller, Klaus Heuner, and Michael Rohrbach for helpful comments.
This work was supported in part by a grant from the Deutsche
Forschungsgemeinschaft (Sonderforschungsbereich 193; Biological Treatment of Industrial Wastewaters) to U.B.G.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie und Hygiene, Universitätsklinikum
Charité, Dorotheenstr. 96, 10117 Berlin, Germany. Phone:
49-30-20934715. Fax: 49-30-20934703. E-mail:
ulf.goebel{at}charite.de.
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Applied and Environmental Microbiology, February 2000, p. 549-557, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
