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Articles |
a Author for correspondence. Fax 61-2-8396-5811; e-mail atodd{at}medau.jnj.com
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Methods: The strategy involves in vitro amplification of genetic sequences using a DzyNA primer that harbors the complementary (antisense) sequence of a 10-23 DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluorescence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts.
Results: Experiments using K-ras plasmid as template demonstrated that DzyNA-PCR allows quantification of DNA over at least six orders of magnitude (r = 0.992). Studies with human genomic DNA demonstrated the ability to resolve as little as twofold differences in the amount of starting template. DzyNA-PCR allowed the detection of 10 or fewer copies of the target. The clinical utility of the assay was demonstrated using DzyNA-PCR to analyze DNA that was isolated from human serum.
Conclusion: DzyNA-PCR is a simple, rapid, and sensitive technique for homogeneous amplification and quantification of nucleic acids in clinical specimens.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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This study describes the use of 10-23 DNAzymes to facilitate the
detection of the products of in vitro amplification by PCR
(4)(5). The strategy, which is known as
DzyNA-PCR, is illustrated in Fig. 1
. PCR is performed using a DzyNA
primer that contains a target-specific sequence and the complementary
(antisense) sequence of a 10-23 DNAzyme. During PCR, amplicons are
generated that contain both target sequences and active (sense) copies
of DNAzymes. A DNA/RNA chimeric reporter substrate, containing
fluorescence resonance energy transfer fluorophores incorporated on
either side of a DNAzyme cleavage site, is included in the PCR mixture.
Cleavage of this reporter substrate produces an increase in
fluorescence that is indicative of successful amplification of the
target gene or transcript. DzyNA-PCR is a generic and flexible strategy
that provides an alternative to other homogeneous amplification and
detection systems, including the TaqManTM
(6), Molecular Beacon (7),
SunriseTM primer (8), and HybProbe
assays (9).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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reporter substrates
The DzyNA reporter substrate SubDz2
(5'-CCACTCguATTAGCTGTATCGTCAAGCCACTC-3') is a chimeric oligonucleotide
containing both RNA (lower case) and DNA bases. The substrate is
designed such that the bond between the GU ribonucleotides is cleaved
by active DNAzymes generated during DzyNA-PCR. Two versions of the
substrate were synthesized, SubDz2 FAM/TAMRA and SubDz2 FAM/DABCYL,
where the reporter 6-carboxyfluorescein
(FAM)1
was incorporated at the 5' end, and the quencher
6-carboxytetramethylrhodamine (TAMRA) or
4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) was incorporated
internally at nucleotide 10. A 3'-phosphate group was added to prevent
extension by DNA polymerase during PCR. The substrates were synthesized
by Oligos Etc., Inc. (Wilsonville, OR).
dna templates for pcr
The plasmid pCRKM contained the genomic sequence between
nucleotides 84 and 289 of the human cellular c-Ki-ras2
protooncogene, exon 1 (GenBank Locus HUMRASK02, Accession no. L00045)
cloned into the vector pCR2.1 (Original TA cloning kit; Invitrogen).
Plasmid was purified by column chromatography (Qiaprep Spin Plasmid
kit; Qiagen) and used as a reference to establish the calibration curve
used for quantification. The starting copy number was determined from
measurements of the absorbance at 260 nm
(A260), and a range of calibrators
from 107 to 100 copy number
was generated from serial dilutions of plasmid DNA. Genomic DNA was
extracted from the human leukemic cell line K562 and from human serum
specimens according to the cationic polymer protocol (10).
The cell line K562 was obtained from the American Type Culture
Collection (CCL 243). Serum was collected with informed consent
from patients with pancreatic or colorectal cancer who were undergoing
treatment at St Vincent’s Hospital (Sydney, Australia).
amplification and detection
Thermal cycling and monitoring of fluorescence during PCR and data
analysis were carried out using the ABI PRISM®
7700 Sequence Detection System (SDS; PE Biosystems),
MicroAmp® optical 96-well reaction plates, and
MicroAmp optical caps (PE Biosystems). PCR reactions contained
0.4 µmol/L 5K49, 0.06 µmol/L 3K45Dz2, 0.2 µmol/L SubDz2, 8
mmol/L MgCl2, 100 mmol/L each dNTP (dATP,
dCTP, dGTP, and dTTP), and 20 U of rRNasin (Promega Corporation) per
50-µL reaction. Reactions shown in Figs. 2
and 4
contained Gold
buffer and 6 U of AmpliTaq Gold DNA polymerase (PE Biosystems).
Reactions shown in Fig. 3
contained 10 mmol/L Tris-HCl, 75 mmol/L KCl,
pH 8.3 (at 25 °C), as buffer and 3 U of AmpliTaq DNA polymerase (PE
Biosystems) preincubated with TaqStartTM antibody
(Clontech) in the ratio 1:10 according to manufacturer’s instructions.
Thermocycling conditions were as described in the figure legends.
Template DNA samples analyzed by DzyNA-PCR were pCRKM plasmid DNA
(10-fold serial dilutions from 107 to
100 copy number; Figs. 2
and 4
), genomic DNA
isolated from the cell line K562 (31–500 ng; Fig. 3
), and genomic DNA
isolated from the equivalent of 5 µL of human serum (Fig. 2B
). All
reactions were performed in duplicate unless otherwise indicated.
Additional control reactions were performed in parallel and contained
all reaction components with the following changes: no-template DzyNA
control (NTC) mixtures lacked template DNA, and standard PCR control
mixtures lacked the primer 3K45Dz2, which was replaced by the 3K45
control primer.
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data analysis
Sequence detection software (PE Biosystems) was used to monitor
the increase in FAM fluorescence at 530 nm following cleavage of
substrate by amplicons harboring active DNAzymes. A cycle threshold
value (Ct) was determined for each sample corresponding to the cycle
when fluorescence exceeded a defined baseline signal (threshold 
Rn)
within the log phase of product accumulation. A calibration curve was
generated when the log of the copy number was plotted against the Ct
value. Quantification of the amount of DNA in reactions containing
unknown numbers of copies of the K-ras gene was estimated
from the calibration curve. Baseline settings for analysis were in the
cycle number 2–10 range. Analysis using sequence detection software
was performed in the absence of correction for the passive reference
6-carboxy-X-rhodamine because this was not included in the DzyNA-PCR
mixtures.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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). Successful amplification of target DNA, and concomitant
cleavage of the reporter substrate by amplicons containing active
DNAzymes, was indicated by an increase in fluorescent emission of the
FAM reporter (
Figs. 2–4
). The fluorescent signal was monitored at each
anneal/extend step, and quantification of template copy number was
determined using an ABI PRISM 7700 SDS. The 3' primer 3K45 was used in standard PCR control reactions to amplify K-ras amplicons lacking the 10-23 catalytic core. No increase in fluorescent signal above background was observed in these control reactions. This control confirms that the increases observed during DzyNA-PCR are attributable to the presence of DNAzyme sequences within the amplicons and not to hydrolysis of the reporter substrate as a result of either high temperatures or the presence of contaminating RNases. Additional experiments using 32P-labeled substrate confirmed that the mechanism of action of DNAzymes generated during DzyNA-PCR is genuine cleavage of the substrate. The presence of intact and cleaved 32P-labeled substrate was visualized by phosphoro-imagery after separation of fragments by polyacrylamide electrophoresis (data not shown).
characteristics of DzyNA-PCR ASSAY
The linear range, detection limit, specificity,
reproducibility, and accuracy of quantification of the DzyNA-PCR assay
were assessed. To determine the linear range and to develop a
calibration curve for purposes of quantification, 10-fold serial
dilutions of plasmid DNA (pCRKM) consisting of
107–100 target copy number
were analyzed. As shown in Fig. 2
, the dynamic linear range was at least six orders of magnitude,
from 107 to 101
target copy
number. The correlation coefficient of the calibration curve was
consistently 0.99 or greater. The assay was specific for target
DNA as shown by the high threshold cycle numbers (Ct values) of the
NTCs, which were consistently at least 10 cycles greater than those
observed in reactions containing 10 target molecules, as determined in
consecutive assays. The reproducibility of Ct values obtained for a
given number of copies of plasmid DNA was assessed within a single
assay by testing replicate reactions containing
105 or 103 copy number. The
CVs of the Ct values of these replicate measurements were 3.3% (n
= 11), and 2.7% (n = 10), respectively.
Similar calibration curves were generated using 10-, 5-, and 2-fold
serial dilutions of genomic DNA. DzyNA-PCR was shown to be capable of
resolving twofold dilutions of genomic DNA across the range 31–500 ng
per 50 µL (r = 0.980; Fig. 3
). During PCR, the number of amplicons doubles with each cycle;
hence determination of twofold differences in the amount of starting
template corresponds to the theoretical limit of resolution for any
PCR-based assay.
clinical application
DzyNA-PCR was used to estimate the concentrations of circulating
K-ras DNA in the serum of patients with gastrointestinal
malignancies (Fig. 2B
). Serum samples (5 µL) were estimated to
contain between 103 and 104
copies of a single-copy gene (K-ras), indicating that the
concentrations of circulating genomic DNA in these patients were in the
range of 0.8–4 mg/L serum. DzyNA-PCR therefore allowed
quantification of circulating DNA using only small amounts of the
clinical specimens. The ability to detect tumor markers, such as
microsatellite instability, aberrant methylation, or mutated oncogenes,
in serum or plasma may provide a new noninvasive tool for diagnosis,
prognosis, and follow-up of cancer (11)(12)(13)(14). Highly
sensitive techniques such as restriction endonuclease-mediated
selective PCR (10)(15) and methylation-specific
PCR (16), which allow detection of activated alleles
in a 1000-fold excess of wild-type alleles, are likely to be most
compatible with noninvasive diagnostic approaches. DzyNA-PCR can be
used to ascertain whether the amount of circulating DNA extracted is
adequate to ensure that the sensitivity of subsequent analyses by these
techniques is not compromised by the addition of insufficient DNA in
reaction mixtures (17). Furthermore, it is technically
feasible to use restriction endonuclease-mediated selective PCR or
methylation-specific PCR primers in conjunction with DzyNA primers for
direct detection of genetic abnormalities in serum samples.
potential for multiplex DzyNA-PCR
Future work in this laboratory will aim at developing multiple
generic substrates, each of which could be labeled with a different
fluorophore and each of which could be adapted to report on the
presence of specific target amplicons. The simultaneous use of multiple
substrates in a single reaction mixture would allow the development of
multiplex DzyNA-PCR assays. In preliminary work, our initial substrate
(SubDz2) was resynthesized with DABCYL replacing the TAMRA moiety.
DABCYL previously has been exploited as a universal quencher on
molecular beacons (7). In a study by Vet et al.
(18), use of this nonfluorescent quencher allowed the
resolution of four molecular beacons with different fluorophores in a
single reaction mixture with analysis by the ABI PRISM 7700.
The substrate SubDz2 FAM/DABCYL was used in the DzyNA-PCR system
targeting K-ras to resolve 10-fold dilutions from
107 to 100 copy
number (r = 0.995; Fig. 4
). In reactions containing template diluted to contain only a
single target molecule, the presence of K-ras was detected
in some but not all reactions (Fig. 4B
). This is likely to reflect
sporadic sampling of single or small numbers of target molecules.
SubDz2 FAM/DABCYL was more compatible with stringent thermocycling
profiles containing annealing/extension temperatures of 50 °C than
was the SubDz2 FAM/TAMRA substrate. This may be the result of enhanced
stabilization of the secondary structure of the hybridizing arms at the
higher temperature when the DABCYL moiety replaces TAMRA.
factors influencing DzyNA-PCR ASSAY EFFICIENCY
Before this study, investigations into the ability of the 10-23
DNAzyme to cleave substrates had largely been confined to physiological
conditions. The conditions required for PCR vary greatly from the
physiological with respect to a number of conditions such as
temperature, ionic strength, and buffer composition. In DzyNA-PCR,
reaction conditions must be compatible with both amplification by PCR
and cleavage by the 10-23 DNAzyme. A range of suitable conditions was
identified in a series of experiments where the DNAzyme-mediated
cleavage of 32P-labeled substrates was quantified
by phosphoro-imagery (H. Impey et al., manuscript in
preparation). In DzyNA-PCR, the primer concentrations were
shown to affect the efficiency and were adjusted to favor production of
the strand containing the active DNAzymes.
The efficiency of cleavage by 10-23 DNAzymes can be influenced by the length of their hybridizing arms (1). Amplicons produced by the K-ras DzyNA-PCR system described above harbored DNAzymes that hybridized to SubDz2 via one short 6-base arm and one long 25-base arm. In initial experiments using SubDz2 FAM/TAMRA, annealing temperatures of 40 °C were used to promote efficient hybridization of the short arm of the DNAzyme in an effort to ensure efficient cleavage. Subsequent experiments using SubDz2 FAM/DABCYL demonstrated that SubDz2 could be efficiently cleaved during PCR incorporating higher annealing temperature steps (50 °C) and that the quencher moiety used to label the substrate could influence the efficiency. Additional studies in this laboratory have shown that DzyNA-PCR systems that generate DNAzymes with longer hybridizing arms facilitate highly efficient cleavage at temperatures ranging between 50 and 60 °C (H. Impey et al., manuscript in preparation). The inherent catalytic activity of the 10-23 DNAzyme is therefore retained at high temperatures such as those typically used during the annealing phase of PCR. Release of the substrate from the DNAzyme after cleavage is ensured by the high temperature used for the denaturation step in PCR.
feasibility for clinical diagnostics
Homogeneous sealed-tube formats have several advantages over
protocols where amplicons are analyzed separately after amplification.
Closed system methods are faster and simpler because they require fewer
manipulations. Furthermore, such systems eliminate the potential for
false positives associated with contamination with PCR products from
other reactions. In DzyNA-PCR, the reporter substrate and the
corresponding primer sequence encoding the DNAzyme can be generic. As
such, new DzyNA-PCR assays could be developed more rapidly than assays
such as TaqMan (6), Molecular Beacons (7), and
HybProbes (9). These assays depend on internal hybridization
of reporter probes that must be developed for each target nucleic acid.
Theoretically, the sequence encoding a generic DNAzyme could be attached to any primer targeting a gene or transcript. In practice, primers are selected for DzyNA assays on the basis of lack of propensity for formation of catalytically active primer-dimers. Not all primer-dimers are expected to harbor catalytically active DNAzymes because this requires the primers to anneal in such a way as to copy the entire catalytic domain and a substantial portion of the hybridizing arm sequence. This provides an advantage over the Sunrise (8) strategy where all primer-dimers produce increases in reporter fluorescence. When DzyNA-PCR is monitored in real time, increases in the fluorescence attributable to primer-dimers can be clearly distinguished from the fluorescence associated with amplification of target nucleic acids. The Ct values produced by active primer-dimer formation in reactions lacking template DNA are consistently at least 10 cycles greater than those observed in reactions containing 10 target nucleic acid molecules.
DzyNA PRIMER SYSTEMS: COMPATIBILITY WITH ALTERNATIVE
AMPLIFICATION STRATEGIES
The general strategy outlined here is potentially very flexible.
In addition to PCR, several other strategies for in vitro amplification
of nucleic acid sequences have been described. These include strand
displacement amplification (19), which produces DNA
products, and transcription-mediated amplification (20),
which produces RNA products. Theoretically, the catalytic nucleic acid
molecule encoded by a DzyNA primer could be either a DNAzyme if PCR or
strand displacement amplification were used, or a ribozyme (catalytic
RNA enzyme) if transcription-mediated amplification was used to mediate
nucleic acid amplification. Furthermore, in vitro evolution technology
has facilitated the discovery of DNAzymes and ribozymes capable of
catalyzing a broad range of reactions, including cleavage
(1)(21)(22)(23) and ligation of nucleic acids
(24), porphyrin metallation (25), and the
formation of carbon-carbon (26), ester (27), or
amide bonds (28). Therefore, it may be possible to develop
systems for detection of in vitro amplification products where the
reporter substrate is a molecule other than a nucleic acid and/or the
readout of the assay is dependent on a modification other than cleavage
of the substrate.
The discovery of the 10-23 DNAzyme has provided a new tool for use in genetic diagnostics. This study has demonstrated the use of DNAzymes to facilitate homogeneous nucleic acid amplification, detection, and quantification in a real-time fluorescent format. Methods of in vitro nucleic acid amplification have widespread applications in genetics, disease diagnosis, and forensics. The development of rapid, homogeneous systems for amplification and detection are required to facilitate the transfer of molecular diagnostics from the laboratory to the clinic.
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Acknowledgments |
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was conceived by Drs. A.V. Todd, C.J. Fuery, and
M.J. Cairns (International patent application (1999) publication no.
PCT/IB99/00754). |
Footnotes |
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1 Nonstandard abbreviations: FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; DABCYL, 4-(4'-dimethylaminophenylazo)benzoic acid; SDS, sequence detection system; Ct, threshold cycle; and NTC, no-template control. 
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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