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Articles |
Division of Clinical Chemistry, Department of Medicine, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg i. Br., Germany.
a Author for correspondence. Fax 49-761-270 3444; e-mail msnauck{at}med1.ukl.uni-freiburg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: In this report we present a single-tube method for genotyping of the 4G/5G polymorphism that combines both rapid-cycle PCR with real-time monitoring of the amplification process and generation of allele-specific fluorescent probe melting profiles on the LightCyclerTM. Two fluorescently labeled hybridization probes recognizing adjacent sequences in the amplicon were present in the reaction mixture. The shorter detection probe spanned the polymorphic site, perfectly matching the 5G allele. After annealing, the fluorophores were in resonance energy transfer, providing real-time monitoring of the amplification process. At the completion of the PCR, fluorescence was monitored as the temperature increased through the Tm of the probe/product duplex, and a characteristic melting profile for each genotype was obtained.
Results: With this method, 32 samples were genotyped within 30 min without the need of any post-PCR sample manipulation. The genotypes of 100 DNA samples determined with the LightCycler were identical to those obtained with conventional PCR and restriction fragment length analysis.
Conclusion: The genotyping of the 4G/5G polymorphism with the LightCycler is a rapid, reliable method that is suitable for typing both small and large numbers of samples.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PAI-1 is a 50-kDa glycoprotein belonging to the serine protease superfamily (serpins) (5). It inhibits both tissue-type and urokinase-type plasminogen activators and is considered as the primary regulator of plasminogen activation (6). An increased plasma concentration of PAI-1 is a marker for first and recurrent myocardial infarction in young patients (1)(2) and for ischemic events in individuals with preexisting atherosclerosis (7). PAI-1 concentrations relate to the degree and severity of atherosclerotic lesions (8), and PAI-1 may play a role in the pathogenesis of cerebrovascular disease and venous thromboembolism (9)(10).
The genetic and environmental determinants of PAI-1 expression are still incompletely understood. It has been postulated that PAI-1 responses to different stimuli in vivo may be mediated by changes of the rate of gene transcription and that sequence elements within the promoter region control this response. A common single nucleotide insertion/deletion polymorphism in the PAI1 promoter (4G/5G) has been identified 675 bp upstream from the start of transcription. This polymorphism produces two alleles containing either four or five sequential guanosines (G) (11)(12). The 4G/5G polymorphism has been related to circulating PAI-1 concentrations in healthy subjects, young patients with myocardial infarction, and patients with non-insulin-dependent diabetes, with subjects homozygous for the 4G (deletion) allele having the highest PAI-1 concentrations (11)(12)(13)(14). In vitro studies have shown increased gene transcription associated with the 4G allele (11)(12). This provides a potential mechanism for the increase in PAI-1 concentrations with the number of 4G alleles seen in vivo. Therefore, genotyping of this polymorphism may become relevant to risk assessment and to the selection and dosing of patients being treated with anticoagulant and fibrinolytic agents, in particular with tissue plasminogen activator.
We present here a single-step method for genotyping of the 4G/5G polymorphism of the PAI1 gene that uses rapid-cycle PCR and subsequent analysis with resonance energy transfer probes on a thermal cycler with real-time fluorescence monitoring (15). After completion of the amplification process, the fluorescence signal is used to analyze the allele-specific melting behavior of the fluorophore-labeled hybridization probe. This is the first report demonstrating that not only single-base substitutions but also single-base insertions/deletions within PCR products can be readily identified by their characteristic melting behavior by the use of sequence-specific fluorescently labeled oligonucleotide hybridization probes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mutation detection using fluorescence resonance energy transfer
Fluorescence monitoring using hybridization probes is based on the
concept that a fluorescence signal is generated if fluorescence
resonance energy transfer (FRET) occurs between two adjacent
fluorophores (16). The first hybridization probe, which is
labeled with fluorescein as donor fluorophore on its 3' end, can
hybridize in close proximity to a second hybridization probe that is
labeled with the acceptor fluorophore LightCyclerTM Red 640
at its 5' end and is blocked from extension at its 3' terminus. The
fluorescein is excited by a LED light source and transfers energy to
the acceptor fluorophore. The acceptor fluorophore then emits light of
a longer wavelength that can be measured with a photodiode. This
detection strategy allows monitoring of the amplification process on a
per-cycle basis because the intensity of the FRET signal depends on the
amount of specific PCR product generated. Even more important,
genotyping using two hybridization probes is possible with a shorter
"detection probe" that spans the polymorphic site and a longer
"anchor probe" that recognizes an adjacent sequence. The greater
stability of the longer anchor probe causes the detection probe
to melt off the template at a lower temperature so that polymorphic
alleles can be distinguished by the melting temperature
(Tm) of the detection probe.
Continuous monitoring of the fluorescence as the temperature is raised
from annealing to denaturation produces a sharp decrease in
fluorescence when the detection probe dissociates from the template.
design of primers and fluorogenic probes
The primer 5'-AGCCAGACAAGGTTGTTGACAC-3' (nucleotides -741
to -720, relative to the transcription start site) and the primer
5'-CAGAGGACTCTTGGTTTTCCC-3' (nucleotides -628 to -607, relative to
the transcription start site) were used to amplify, respectively, a
134- or a 135-bp fragment of the human PAI1 gene that
harbors the polymorphic site 675 nucleotides upstream from the start of
transcription (GenBank locus HSPAI11) (17). The
amplification primers were synthesized by standard phosphoramidite
chemistry (MWG-Biotech).
The detection probe was a 18-mer oligonucleotide, labeled at the 3' end
with fluorescein. The sequence 5'-TGACTCCCCCACGTGTCC-3' is
complementary to the leading strand of the 5G allele
(underlined nucleotides; Fig. 1
). The anchor probe (5'-ACTCTCTCTGTGCCCCTGAGGGCTCT-3') was a
26-mer labeled with LightCycler Red 640 at its 5' end and modified at
the 3' end by phosphorylation to block extension. The anchor probe
binds at a distance two bases 3' from the detection probe. Both
fluorophore-labeled probes were synthesized and purified by
reversed-phase HPLC by TipMolBiol.
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fluorescence protocol
The analysis was carried out on a LightCycler (Roche Molecular
Biochemicals). PCR was performed by rapid cycling in a reaction volume
of 10 µL with 0.3 µmol/L each primer, 0.2 µmol/L anchor and
detection probes, and 50 ng of genomic DNA. As a reaction buffer, the
LightCycler DNA Master Hybridization Probes buffer from Roche Molecular
Biochemicals was used (cat. no. 2015102). This buffer is provided as a
10-fold stock solution containing nucleotides, Thermus
aquaticus DNA polymerase, and 10 mmol/L
Mg2+. The final Mg2+
concentration in the reaction mixture was adjusted to 4 mmol/L. The
samples were loaded into glass capillary cuvettes (cat. no. 1909339;
Roche Molecular Biochemicals) and centrifuged to place the sample at
the capillary tip before capping. After an initial denaturation step at
94 °C for 45 s, amplification was performed using 50 cycles of
denaturation (94 °C for 0 s), annealing (57 °C for
5 s), and extension (72 °C for 2 s). The temperature
transition rates were programmed at 20 °C/s from denaturation to
annealing, 3 °C/s from annealing to extension, and 20 °C/s from
extension to denaturation. Fluorescence was measured at the end of the
annealing period of each cycle to monitor amplification. After
amplification was complete, a final melting curve was recorded by
cooling the reaction mixture to 45 °C at 10 °C/s, holding it at
45 °C for 3 min, and then slowly heating it to 75 °C at
0.2 °C/s. Fluorescence was measured continuously during the slow
temperature ramp to monitor the dissociation of the fluorescein-labeled
detection probe. The fluorescence signal (F) was plotted in
real time against temperature (T) to produce melting curves
for each sample (F vs T). Melting curves were
then converted to derivative melting curves by plotting the negative
derivative of the fluorescence with respect to temperature against
temperature [-(dF/dT) vs T]. The
entire process took ~30 min with no separate manipulation of the
product necessary.
genotyping by allele-specific restriction enzyme site analysis
For confirmation of genotypes, allele-specific restriction enzyme
site analysis (ASRA) was performed as described previously
(18). This protocol uses a modified upstream primer
producing a BslI cutting site with the 5G allele
but not with the 4G allele. In brief, a 99- or 98-bp
fragment, depending on the promoter allele present, was amplified from
genomic DNA using the primers described by Margaglione et al.
(18). Amplifications were performed in volumes of 50 µL
containing 100 ng of genomic DNA, 0.8 µmol/L each primer, 200
µmol/L each deoxyribonucleotide, 10 mmol/L Tris-HCl, pH 8.3, 1.5
mmol/L MgCl2, 50 mmol/L KCl, 0.1 g/L gelatin, and
1 U of Thermus aquaticus DNA polymerase (Roche Molecular
Biochemicals). The protocol included an initial denaturation step at
94 °C for 2 min, followed by 40 cycles of 20 s of denaturation
at 94 °C, 20 s of annealing at 60 °C, and 20 s of
elongation at 72 °C. Thermocycling was carried out on a conventional
programmable thermocycler (UNO II; Biometra), followed by a final
elongation step of 72 °C for 5 min. Aliquots (10 µL) of the PCR
mixture were then digested for 4 h at 55 °C with 5 U of
BslI, using the restriction buffer recommended by the
manufacturer (New England Biolabs). The resulting fragments (77 and 22
bp for the 5G allele, and 98 bp for the 4G
allele) were electrophoresed on 3.5% GTG agarose (FMC Bioproducts),
visualized with 0.5 mg/L ethidium bromide, and examined under
ultraviolet illumination.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. When fluorescence was measured at the end of
each annealing phase, the fluorescence signal increased as product
accumulated. Under our conditions, the fluorescence signal appeared
above background after 20 cycles (Fig. 2
). No increase in fluorescence signal was observed in the
absence of template (Fig. 2
, curve D).
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The process of hybridization and melting of the detection probe to the
target was monitored by melting curve analysis. Melting of the sample
homozygous for the 4G allele produced a rapid decrease in
fluorescence at 56–57 °C. In contrast, in the sample homozygous for
the 5G allele, the transition occurred at 63–64 °C. The
heterozygous sample exhibited two distinct decreases in fluorescence,
corresponding to the presence of amplicons derived from both alleles.
Peaks were obtained at the respective melting temperatures by plotting
the negative derivative of the fluorescence signal with temperature vs
temperature [-(dF/dT) vs T].
Accordingly, the melting peak of the sample homozygous for the
5G allele was at 64 °C (Fig. 3
, curve C), whereas in the sample homozygous for the
4G allele, a melting peak was obtained at 57 °C (Fig. 3
, curve A). The heterozygous sample contained both types of
targets and thus generated both peaks (Fig. 3
, curve B). With different
samples showing different amplification efficiencies, the derivative
melting curves were highly reproducible, with melting peaks differing
by <1.5 °C for the same allele, allowing easy and unambiguous
assignment of genotypes to the respective melting curves.
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To evaluate the reliability of the fluorescence genotyping, 100 human
DNA samples were genotyped for the 4G/5G
polymorphism by both the primer-mediated ASRA and the fluorescence
method. The PCR products and fragments obtained by ASRA were of the
expected sizes (Fig. 4
), and the genotypes determined with both methods were in 100%
concordance. Thirty-two samples were homozygous for the 4G
allele, 49 were heterozygous, and 19 were homozygous for the
5G allele. The genotyping of the 100 samples on the
LightCycler was completed within 2 h, whereas the ASRA protocol
took 8 h and required several manual sample processing steps.
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Comparison of the costs for reagents and disposable material showed that both methods are about equally expensive, with costs of approximately $2.00 (US) per typing reaction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because there is some evidence that PAI-1 release from atherosclerotic lesions is increased as part of an acute-phase response, it is also possible that the increased PAI-1 seen in several studies may result from, rather than lead to, atherosclerosis or thrombosis (21). Furthermore, an association between PAI-1 concentrations and coronary artery disease may be concealed by the close correlation between PAI-1 concentrations and other established risk factors, such as insulin resistance (22)(23). Furthermore, measurement of PAI-1 activity and PAI-1 antigen is complicated by several preanalytical requirements. For example, rapid sample handling and quick freezing of the samples is necessary because PAI-1 is a labile molecule with a half-life of functional activity of ~4 h under physiological conditions (6). If PAI-1 antigen is to be measured, precautions must be taken to avoid release of PAI-1 from platelets. Generally, the time for blood sampling must be standardized because of the diurnal fluctuation of fibrinolytic activity (24). Therefore, the association of the 4G allele with increased plasma PAI-1 might be more reliable and robust as a predictor of the thrombotic risk than plasma concentrations. In addition, in the general population, plasma concentrations have a continuous distribution, which makes it difficult to establish cutoff values that can identify individuals at risk.
The growing evidence that the PAI-1 concentrations in the circulation are related to the common 4G/5G polymorphism in the promoter of the PAI1 gene has opened the possibility of using PAI1 genotype as a marker for life-long exposure to differing PAI-1 concentrations. Furthermore, the determination of the PAI1 polymorphism based on the PCR is simple in terms of preanalytical precautions and requires only a small number of nucleated cells.
PCR with allele-specific oligonucleotide hybridization (11)(14)(25), amplification with sequence-specific primers (26)(27)(28)(29), ASRA (18)(30), and single-strand conformation polymorphism (31) have been used to genotype the PAI1 promoter polymorphism. PCR with allele-specific oligonucleotide dot-blot hybridization is time-consuming, and the conditions for allele-specific hybridization based on a single-base deletion/insertion are crucial. The method may be affected by variations of the quality of the PCR products and is sensitive to small changes of the hybridization conditions. PCR with sequence-specific primers is an increasingly popular method for genotyping biallelic systems. However, most protocols to date require the use of separate tubes for specifically amplifying each allele, adding time and expense. In addition to the inherent risk of false-positive amplification, the performance of the sequence-specific primers must be verified by the amplification of internal control fragments. Techniques based on ASRA need PCR conditions of sufficient specificity to produce a clean amplification product that can be enzymatically digested and analyzed by electrophoresis. Furthermore, the procedure may be complicated by incomplete digestion of the amplicons.
The potential benefits of homogeneous detection systems have long been recognized (32), and recently several fluorescence-based methods for typing biallelic systems have been described that improve sample throughput (33)(34)(35)(36)(37). In the present study, we used probe hybridization and FRET to monitor product accumulation during rapid-cycle DNA amplification and to determine PAI1 promoter genotypes by melting-curve analysis of the amplified fragment on the LightCycler. This protocol allows fluorescence genotyping of 32 samples in <30 min without any need for enzyme digestion or electrophoresis. Because this method is performed in a closed system with no postamplification processing, potential problems with sample tracking errors and end-product contamination are eliminated. The robustness and reliability of the fluorescence genotyping was well documented by the complete concordance of 100 genotypes determined with both the conventional restriction fragment length analysis and the LightCycler protocol.
This new method combines simple sample processing and rapid analysis; it therefore affords both high-throughput genotyping and rapid results. Furthermore, because hands-on time is short, this method permits PAI1 genotyping in a very economical manner. Compared with published fluorescence protocols applied on the ABI 7700 (35)(36) or the ABI 5700 from Perkin-Elmer (37), the analytical runs on the LightCycler are about three times faster. On the other hand, the instruments from Perkin-Elmer allow the simultaneous processing of three times as many samples. Although the sample throughput of both systems is similar, the faster processing of the LightCycler allows more flexibility in the use of the instrument.
Similar to the conventional use of allele-specific oligonucleotide probes in mutation detection, melting curve analysis depends on the thermal stability difference between matched and mismatched DNA duplexes. Many years ago, Wallace et al. (38) showed that single-base mismatches lowered the melting temperature of oligonucleotide probes by several degrees Celsius. The extent of destabilization depends on the duplex length, the particular mismatch, the nearest neighbor environment, and the position of the mutation site relative to the probe (39). The molecular characteristics of the 4G/5G polymorphism is different from single-base mismatches in that a single guanosine is inserted or deleted, giving rise to alleles with four or five guanosines, respectively. By using an 18-mer detection probe that is fully complementary to the leading strand of the 5G allele, we achieved a Tm shift between both alleles of 7.0 °C. This difference in Tm between homoduplex and heteroduplex is in the range reported for single-base mismatches in 18-mers (40). This indicates that the design of our detection probe was appropriate to discriminate between genotypes at that locus. Most likely, by annealing to the 4G allele, the hybridization probe is forced into a bend, with one cytosine being unable to hybridize and weakened hydrogen bonds in its neighborhood.
Our results also suggest that other base insertion/deletion polymorphisms with potential relevance to human disease can be conveniently genotyped with a homogeneous assay using fluorescence-labeled hybridization probes (41)(42).
To date, three single-step genotyping methods using the LightCycler have been described. The first two applications, designed for detection of the factor V Leiden mutation and the C677T point mutation in the methylenetetrahydrofolate reductase gene, used one Cy5-labeled primer and one fluorescein-labeled hybridization probe with asymmetric amplification of the target sequence. In contrast, Bernard et al. (43) in a more recent work developed an assay for homogeneous multiplex genotyping of hemochromatosis mutations that uses two pairs of hybridization probes binding to internal sequences of two fragments amplified simultaneously. We used a similar approach by also using a pair of fluorophore-labeled probes that hybridize to the same strand internal to an unlabeled primer set. The results of Bernard et al. as well as ours document that an internal hybridization probe pair works equally well. Because the synthesis of labeled primers is known to be more difficult than the end-labeling of internal probes, the probe pair system may be more flexible and cost-effective.
In summary, we developed a homogeneous (closed tube) assay for rapid genotyping of the 4G/5G promoter polymorphism of the PAI1 gene on the LightCycler that allows both real-time monitoring of the amplification process and allele-specific analysis of the probe melting curves at completion of the amplification. Genotyping of 32 samples is accomplished within 30 min, making this method ideally applicable to PAI1 typing in both small and large numbers of patients.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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