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Technical Briefs |
1-Antitrypsin Deficiency Alleles Pi*Z and Pi*S by Real-Time Fluorescence PCR and Melting Curves
1
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef Strauss Allee 11, 93042 Regensburg, Germany;
2
Department for Clinical Chemistry, University Hospital Freiburg, 71106 Freiburg, Germany;
a author for
correspondence: fax 49-941-944-6202, e-mail Charalampos.Aslanidis{at}klinik.uni-regensburg.de
Protease inhibitor 1
(
1-antitrypsin; AT) is the main serum
inhibitor of proteolytic enzymes. In AT deficiency, enzymes such as
neutrophil elastase can damage the lung tissues, leading to pulmonary
emphysema. More than 90 different alleles have been identified to date
for the protease inhibitor 1 gene (PI). The three most
important variants are type M (90% of population), type S
(PiS), and type Z (PiZ), of which types S and Z
are two of the more common abnormal variants. Homozygotes of
type Z have a considerable reduction in the serum AT concentration and
may develop pulmonary emphysema or hepatic cirrhosis. SZ-heterozygotes
are less severely affected (1)(2)(3). The allele frequencies of
the most common alleles, PiS and PiZ, are
0.02–0.04 or 0.01–0.02, respectively, with 0.2% of the
general population being homozygous for either the
PiZ or PiS allele. Typically, different
phenotypes have been characterized by isoelectric focusing.
PCR-based technologies are now being used widely for the identification of the mutations underlying the PiZ allele (Glu342Lys, GAG to AAG) and the PiS allele (Glu264Val, GAA to GTA), thus simplifying mutation detection (4). Although these methods give rise to unequivocal results, they are time-consuming and require optimization of the PCR reaction to eliminate nonspecific PCR products that would disturb the genetic analysis. Recently, a new detection methodology based on hybridization of amplicon-specific oligonucleotides with adjacent fluorophores capable of fluorescence resonance energy transfer was introduced and was used in a new high-speed thermal cycler (LightCyclerTM; Roche Diagnostics) that uses glass capillaries and hot air for heating (5). This technology enables the real-time detection of the specific PCR product followed by detection of the mutation by identification of the melting behavior of one of the two hybridization oligonucleotides (6). To this end, one hybridization primer matches the wild-type sequence (or mutant sequence), with the variable nucleotide approximately in the middle of the sequence, and has LC-red640 as the fluorophore at its 5' end (detection probe, phosphorylated at 3' end); a second hybridization primer (anchor primer) is located upstream at a distance of 1–3 nucleotides and is labeled with fluorescein at its 3' end. During the cycling process, the hybridization probes hybridize the specific PCR product at the annealing temperature and fluorescence is generated and monitored. Cycling times are kept very short because of quick adaptation of the temperature in the glass capillary (high surface-to-volume ratio) and very fast temperature transition rates (20 °C/s). DNA is denatured instantly when the temperature in the glass capillary has reached 94 °C; therefore, no additional incubation time is required before temperature is reduced to the annealing temperature (setting is 94 °C for 0 s). After the PCR is completed, the PCR mixture is denatured and the temperature is lowered to 40 °C to enable binding of the hybridization probes to all PCR products (maximum fluorescence) and slowly raised to 80 °C to permit melting of the detection probe, which is monitored by the decline of the fluorescence. Melting curves are converted to melting peaks, allowing easy distinction of the wild type from the mutant. The whole process is completed within 30 min. We have used this high-speed mutation detection successfully for the identification of the apolipoprotein E isoforms, the apolipoprotein B3500 mutation, the prothrombin G20210A mutation, and the C677T mutation in methylenetetrahydrofolate reductase (7)(8).
In this study, we describe mutation detection in the PI 1
gene to identify the PiZ and PiS alleles, using
the LightCycler technology. We used genomic DNA isolated from
EDTA blood from individuals that had been typed previously by
PCR-restriction fragment length polymorphism (RFLP) analysis. The
primer sequences for PCR and the sequences of the hybridization
probes are shown in Table 1
. Primers ATS0 and ATS2 were used for amplification of a 238-bp
region for identification of the PiS allele. The primers
for the characterization of the PiZ allele were ATZ0 and
ATZ2 and generated a 253-bp PCR product from genomic DNA. The
3'-phosphorylated detection primer for the PiS allele was
AATSM and was located downstream of the anchor primer AATSA at a
distance of two nucleotides. The detection primer for the
PiZ allele was AATZM, and the corresponding anchor primer
was AATZA, located upstream with a distance of three nucleotides.
Anchor and detection primers were in the sense orientation.
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PCR and melting curve determinations were performed in 20-µL volumes in glass capillaries (Boehringer Mannheim). For PiS characterization, the following pipetting scheme was used: 10.4 µL of H2O, 1.6 µL of 25 mmol/L MgCl2, 1 µL of ATS0 and 1 µL of ATS2 at 5 pmol/µL each, 1 µL each of AATSA and AATSM from 4 pmol/µL stock solutions, and 2 µL of DNA-Master Hybridization Probes (Roche Diagnostics), containing Taq polymerase, reaction buffer, a dNTP mixture, and 10 mmol/L MgCl2 as a 10x concentrate. For PiZ allele identification, 11.2 µL of H2O, 0.8 µL of 25 mmol/L MgCl2, 1 µL of ATZ0 and 1 µL of ATZ2 at 5 pmol/µL each, 1 µL each of AATZA and AATZM from 4 pmol/µL stock solutions, and 2 µL of DNA-Master Hybridization Probes were added in a 20-µL reaction. Genomic DNA (2 µL, 50–200 ng) was used for amplification. Fluorescently labeled hybridization probes were synthesized by TIB MOLBIOL (Berlin). Cyclingconditions were identical for both applications, with initialdenaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 0 s, annealing at 55 °C for 10 s, and extension at 72 °C for 15 s, with a ramping rate of 20 °C/s. Fluorescence was monitored at the end of each 10-s annealing phase. After the amplification, melting curves were generated by denaturation of the reaction at 94 °C for 0 s, holding the sample at 40 °C (35 °C for PiZ) for 5 s, and then slowly heating the sample to 80 °C (70 °C for PiZ), with a ramp rate of 0.2 °C/s, and simultaneously monitoring the decline in fluorescence. Melting curves were converted to melting peaks by calculating the negative derivative of the fluorescence with respect to temperature (-dF/dT) against temperature.
The fluorescence profiles generated from DNA samples and negative
controls are shown in Fig. 1
. When the hybridization probes for the PiS allele
were used (Fig. 1A
), fluorescence increased constantly in the samples
with the DNA (samples 2 and 3), whereas no fluorescence was detected in
the H20 control (sample 1). Analysis of PCR
products on agarose gels revealed the presence of the specific 238-bp
PCR product (Fig. 1C
). The melting curves of the same samples are shown
in Fig. 1B
. The melting point (Tm) of
the wild-type sample (curve 3) was at 55.7 °C, whereas the
heterozygous sample (curve 2) produced two melting peaks at 48.6 °C
and 55.7 °C. Despite the equal amounts of PCR product in the
capillaries, the fluorescence intensity in sample 2 was substantially
lower than in sample 3. This is because the
Tm of the mismatched probe in one-half
of the PCR products from the heterozygotes (sample 2) was below the
annealing temperature in the PCR reaction. Therefore, when homozygous
mutant individuals are analyzed, no fluorescence is generated during
the PCR. However, melting peaks will reveal the proper genotype because
melting curves start at a low temperature.
|
The fluorescence monitored with the hybridization probes specific for
the PiZ allele is illustrated in Fig. 1D
. The three DNA
samples of different genotypes generated different fluorescence
signals, although the analysis of the PCR products on agarose gels
revealed equal amounts of PCR product (Fig. 1F
). The detection probe
AATZM was derived from the wild-type sequence and matched the
non-mutant PCR products perfectly, leading to stable hybridization at
the annealing temperature, whereas base pairing with PCR products from
homozygous mutant individuals (curve 4, Fig. 1D
) was impaired because
of a low Tm (55.8 °C) compared
with a higher Tm in the wild-type
sample (curve 3), which was 61.5 °C (Fig. 1
E).
To date, we have analyzed >50 individuals for the PiS and
PiZ alleles. The results were consistent with the results
from PCR-RFLP analyses. The basis for the design of the PCR primers is
the same as required for traditional thermal cyclers and has been
described in detail elsewhere (7)(8).
Time-consuming optimization procedures are not required. An additional
advantage of this technology is that contaminating, nonspecific PCR
products do not affect the results. A distance of two nucleotides
between the anchor primer and the detection primer was ideal for
efficient energy transfer and signal generation. The hybridization
probes for the PiS allele (anchor and detector primer) had
a distance of two nucleotides, whereas the probes in the
PiZ application were at a distance of three nucleotides. It
is conceivable that the difference in signal intensity seen in Fig. 1
, A and D (5 vs 1.7) results from a less efficient energy transfer at a
distance of three nucleotides.
In our opinion, this methodology is more accurate than conventional, gel-based assays because it facilitates the identification of the gene-specific PCR product and uses this product for mutation detection. In a traditional PCR-RFLP-based method, contaminating PCR products would scramble the analysis. Therefore, this high-speed, small-volume alternative, which generates lower reagent costs and considerably lower labor costs, is a highly competitive technology in a routine laboratory.
We have established high-speed (30 min) and easy to perform mutation detection for the alleles PiS and PiZ of the gene for protease inhibitor 1, which is responsible for AT deficiency, by use of the LightCycler technology and melting curves. This system minimizes PCR contamination attributable to sample handling (closed capillaries throughout the PCR and detection processes) and does not require digestion of PCR products with restriction enzymes and/or fragment separation on gels.
References
-1-antitrypsin gene and its deficiency states. Trends Genet 1989;5:411-417.
[ISI][Medline]
[Order article via Infotrieve]
-1-Antitrypsin deficiency, emphysema, and liver disease. Genetic basis and strategies of therapy. J Clin Investig 1990;85:1343-1352.
1-Antitrypsin deficiency in Europe: geographical distribution of Pi types S and Z. Respir Med 1998;92:367-377.
[ISI][Medline]
[Order article via Infotrieve]
1-proteinase inhibitor deficiency alleles Pi*Z and Pi*S by DNA analysis. Eur J Clin Chem Clin Biochem 1996;34:761-764.
[ISI][Medline]
[Order article via Infotrieve]
A 20210 and methylenetetrahydrofolate reductase CT 677 mutation detection using real-time fluorescence PCR and melting curves. Biotechniques 1999;27:234-238.
[ISI][Medline]
[Order article via Infotrieve]
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