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Articles |
1-Antitrypsin Genotyping by Multiplex Real-Time Fluorescence PCR with the LightCycler
1
Department of Clinical Chemistry, Georg-August-University, Robert Koch Strasse 40, 37075 Goettingen, Germany.
a Author for correspondence. Fax 49-551-39-2955; e-mail nahsen{at}gwdg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-Antitrypsin is the major plasma
serine protease inhibitor. Its deficiency is mainly associated with the
alleles PI*S and PI*Z and can
lead to obstructive lung disease in adults and to liver cirrhosis
during childhood. Methods: A multiplex PCR method has been established that uses two sets of primers to amplify the gene regions covering the PI*S or PI*Z mutations sites. Mutation detection was performed on the LightCycler by melting curve analysis of detection probes labeled with two different fluorescent dyes, LC-Red640 and LC-Red705.
Results: Unequivocal genotyping results were obtained for all investigated samples in an assay time of ~30 min. The color compensation procedure greatly improved the readability of the resulting diagnostic melting curves.
Conclusions: To our knowledge, this is the first report of
simultaneous detection of two mutations in a single tube by PCR of
genomic DNA and the use of two different reporter dyes with the
LightCycler color compensation feature. This approach is a rapid,
convenient, and economic alternative to other methods described to date
for the detection of 1-antitrypsin deficiency
alleles.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin (1AT), the major plasma
serine protease inhibitor. The protein deficiency is caused by improper
intracellular protein aggregation and secretion or by increased
intracellular protein degradation mainly attributable to two mutant
alleles, designated PIZ and PIS (1). Compared with the wild-type PIM allele, the
PIS allele is characterized by an A
T exchange in exon 3
that codes for a change of glutamine to valine at position 264 of the
protein (2). The PIZ genotype is caused by a
GA mutation in exon 5 that codes for a glutamine-to-lysine exchange
at position 342 of the protein (3). 1AT deficiency can
lead to development of liver cirrhosis during childhood, or more often
to development of obstructive lung disease in adults caused by an
imbalance in the protease/protease inhibitor system with concurrent
overactivity of neutrophil elastase. Patients with clinical
manifestation of the disease usually carry the homozygous
PIZZ genotype and have substantially reduced 1AT plasma
concentrations. Patients with PISZ or PIMZ
genotypes have less reduced 1AT concentrations and usually manifest
obstructive lung disease only if they are smokers. The genetic variants
of the 1AT protein have been identified by isoelectric focusing, a
laborious method that often gives equivocal results and
therefore requires repeated testing (4). Both the
PIS and PIZ alleles are rare or absent in black
or oriental populations but can be as frequent as 1.5–2.9% and
0.4–2.3%, respectively, in different white populations
(4). Apart from these deficiency-associated alleles, other
rare alleles, including null alleles, have been reported. Such alleles
must be considered in patients with low concentrations of 1AT
protein and absence of the PIS or PIZ alleles.
The throughput of genotyping methods can be substantially improved by
multiplex PCR, with a concurrent reduction in the use of reagents and
DNA. Detection of 1AT genotypes is ideally suited to such a
procedure because both deficiency alleles, PIS and
PIZ, must be taken into account. This has been accomplished
previously by the use of conventional PCR methods (5)(6). The LightCycler (Roche Diagnostics) is a
rapid PCR cycler with an integrated three-channel fluorescence
photometer that eliminates the need of any postamplification sample
processing. Oligonucleotide probes labeled with different dyes are used
for mutation detection. Probes added to a PCR reaction mixture will
specifically hybridize to their complementary strand. Mutations under
the probe decrease the stability of this duplex and lead to decreased
melting temperatures (7). This principle of mutation
detection has already been used for factor V (8),
methylenetetrahydrofolate reductase (9), prothrombin G20210A (10), and hemochromatosis (11) genotyping. When
two probes in the assay hybridize adjacent to each other, fluorescence
resonance energy transfer (FRET) occurs between the fluorescein on the
detection probe and the LC-Red dye (Roche Biochemica) on the anchor
probe, producing a specific fluorescence emission. FRET is stopped when
the detection probe melts off the strand. A mutation under the probe
causes a lower probe melting point. Primers internally labeled with the
reporter dye can also serve as counterparts of the fluorescein
detection probe. Although the emission spectra of most dyes overlap,
the LightCycler can be calibrated in such a way that the software
virtually eliminates the crossover of one dye into the detection
wavelength of the other.
The aim of this study was to establish a multiplex PCR method for PIS and PIZ genotyping by use of two different reporter dyes and to show that the color compensation procedure can greatly improve the readability of the melting curve analysis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A), four oligonucleotides were used in the assay, of which two
were the labeled anchor and detection probes and two served as
amplification primers. In the case of the AAT-Z PCR (Fig. 1B
), three
oligonucleotides were used with an internally labeled primer and a
labeled detection probe.
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For the genotyping of the 1AT S and Z
alleles (GenBank accession nos. K02212 and J02619), new primer sets
were constructed:
For PIS genotyping, the primers and probes were as follows:
the forward primer (AAT-S-for) was 5'-AGC GTT TAG GCA TGT TTA AC-3',
and the reverse primer (AAT-S-rev) was 5'-AAG TTT ATA CAG AGT AGC AGT
G-3'. The mutation site was covered by a PIS genotype
complementary detection probe: AAT-S-probe (5'-CAC CTG GTA AAT GAA
CTC-3'-FLU) labeled with fluorescein as indicated (Fig. 1A
). The 3' end
of the AAT-S-anchor probe was phosphorylated to prevent probe
elongation by the Taq polymerase. The adjacent anchor probe
AAT-S-anchor (5'-LC-Red705-CCC ACG ATA TCA TCA CCA AGT TCC-3'-PHO) was
5' labeled with the LC-Red705 dye, which exhibits an emission maximum
at 705 nm.
For PIZ genotyping, the primers and probes were as follows:
the forward primer (AAT-Z-for) was 5'-GTG CAT AAG GCT GTG
CTG AC-3', and the reverse primer (AAT-Z-rev) was 5'-GGT
GGG ATT CAC CAC TTT TC-3'. A thymidine amino-modifier (dT
aminomodifier; Glen Research) was incorporated at the underlined
position in the AAT-Z-for primer. The dye LC-Red640 was linked to the
amino-modifier by reaction with the LC-Red640-N-hydroxysuccinimide
ester and purified according to the manufacturer’s instructions. The
emission maximum wavelength of this dye is at 640 nm. The mutation site
was covered by a PIZ genotype complementary detection
probe, AAT-Z-probe (5'-GCT TCA GTC CCT TTC TTG TCG A-3'-FLU), labeled
with fluorescein as indicated (Fig. 1B
).
color compensation procedure
The color compensation procedure was performed according to the
manufacturer’s instructions (LightCycler color compensation set; Roche
Biochemica). After activation of the software’s color compensation
option, it was possible to read the results from two different
analytical PCRs in one tube. In the PIZ PCR, the LC-Red640
dye was used and the result was detected in channel two, whereas in the
PIS PCR, the fluorescence of the LC-Red705 dye was visible
in channel three.
genomic dna extraction
Genomic DNA was extracted using a commercial resin-based method
(QIAamp DNA blood kit; Qiagen) or by a simple method modified according
to Rudbeck and Dissing (12), which takes <10 min. In brief,
5 µL of anticoagulated blood was added to 1 mL of PCR-grade water in
a 1.5-mL microcentrifuge tube. The tube was immediately centrifuged (12
000g for 1 min), and the supernatant was removed. The
remaining leukocyte pellet was lysed by the addition of 20 µL of 0.2
mol/L NaOH, vortex-mixed thoroughly, and incubated for 5 min. Neutral
pH was restored by the addition of 180 µL of 0.04 mol/L Tris puffer,
pH 7.5, containing 50 g/L Chelex-100 resin, biotechnology grade
(Bio-Rad). The DNA solution can be stored at 4 °C for at least 6
months. Neither the choice of anticoagulant (EDTA, heparin, or citrate)
nor the freezing of samples before DNA isolation affected the method or
inhibited PCR amplification.
multiplex pcr protocol
PCR reactions were carried out in a final volume of 20 µL in
LightCycler glass capillaries. The reaction mixture consisted of 1 µL
of DNA solution, 1 U of Taq DNA polymerase (Boehringer Mannheim), 2
µL of 10x PCR buffer (Boehringer Mannheim), 0.2 mmol/L each dNTP
(Boehringer Mannheim), 2.5 mmol/L MgCl2, 500 mg/L
bovine serum albumin (New England BioLabs), and 50 mL/L dimethyl
sulfoxide (Sigma). Amplification primers were added at different
concentrations to account for the different PCR efficiencies and to
ensure adequate product formation from both primer sets: for the AAT-S
PCR, 0.25 µmol/L AAT-S-for, 0.25 µmol/L AAT-S-rev, 0.1 µmol/L
AAT-S-probe, and 0.3 µmol/L AAT-S-anchor; for the AAT-Z PCR, 0.3
µmol/L AAT-Z-for, 0.3 µmol/L AAT-Z-rev, and 0.1 µmol/L
AAT-Z-probe. PCR-grade water was added to the final volume of 20 µL.
Each set of PCRs included a heterozygous DNA control, a contamination
control from the DNA preparation, and a water control. The
contamination control was prepared by mixing all of the reagents for
DNA isolation but excluding DNA, and would indicate
contamination of reagents used for the DNA isolation in contrast to
reagents used in the PCR itself, where possible contamination would
show up in the water control. The genotyping of control DNA was
performed by a restriction fragment length polymorphism-PCR
(6). The fluorometer gain setting was 5 in channel 1, 10 in
channel 2, and 30 in channel 3. The cycling program consisted of
initial denaturation for 30 s at 95 °C, followed by 55 cycles
of 95 °C for 0 s, 50 °C for 5 s, and 72 °C for
5 s, with maximum ramp rate. The program for analytical melting
was 95 °C for 30 s, 40 °C for 30 s, and an increase to
70 °C at a 0.1 °C/s ramp rate. Amplification and detection
occurred in the same closed tube in <40 min. The PCR conditions and
cycler program were essentially the same as those used previously (10)(13),
thereby fully integrating the 1AT
genotyping into our single master mixture, single cycler program
approach for diagnostic genotyping.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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If on-line PCR monitoring is required, it is advisable to monitor the fluorescence in channel two. Because the melting point of the AAT-Z probe is higher than that of the AAT-S probe, this produces a better hybridization fluorescence signal at the acquisition temperature. During the analytical melting step, fluorescence is measured simultaneously in all three channels of the LightCycler. The concentrations of the primers for the multiplex PCR were optimized to give well-shaped melting curves for all investigated alleles. This ensures that both PCR reactions are amplified to a similar extend. The PCR products were also visualized by agarose gel electrophoresis after ethidium bromide staining. Whenever a good FRET signal occurred in both fluorescence channels, distinctive bands were observed by agarose gel electrophoresis.
Successful amplification was evident from the appearance of specific
fluorescence and the display of derived melting curves. In the case of
a homozygous mutation, there was no mismatch under the
mutation-compatible probe and a single melting peak with a
characteristic high temperature was seen. A single-base-pair-mismatched
probe, i.e., wild-type DNA, caused strand instability and consecutive
melting at a lower temperature. The result was a single melting peak at
a characteristic lower temperature. Patients with heterozygous
mutations accordingly showed two melting peaks. The empirical melting
points determined by the LightCycler software (Software Package V3.1
Data Analysis Module) were ~62.5 °C for PIZ alleles,
55.0 °C for PIS alleles, 59.5 °C for the wild type at
the site of the PIZ allelic mutation, and 49.5 °C for
the wild type at the site of the PIS allelic
mutation. Typical melting curves resulting from genotyping with
this method and active color compensation are illustrated in Fig. 2
. It is noteworthy that the color compensation works
independent of the type of assay (three vs four oligonucleotides) and
coupling of the dyes. When the same samples were analyzed with a color
compensation file that had been calibrated 8 weeks earlier, the
emission crossover of LC-Red640 into channel 3 (705 nm) was not
sufficiently suppressed (Fig. 3
, A and B). If analysis was performed without color compensation
(Fig. 3
, C and D) the substantial crossover of LC-Red640 into channel 3
was even more evident. Reading of the melting curves may be misleading
because of overlaps in both the melting points and the emission
spectra.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Multiplex PCR with hybridization probes has been used before on the
LightCycler for hemochromatosis genotyping (11), but it was
performed with two probes labeled with the same dye. This is possible
only if the different melting points of the two probes are always
clearly discernible, no matter what genotype is present under each
probe. A second reporter dye, LC-Red705, is now available for the
LightCycler, and its fluorescence is read in a separate channel. It is
a general feature of fluorescent dyes that they exhibit broad emission
spectra. This leads to crossover of LC-Red640 emission into the
detection wavelength of LC-Red705 (Fig. 3
, B and D). The application of
color compensation on the LightCycler to correct this situation was
demonstrated recently using a model system with artificial templates (16).
We showed here that the compensation is also possible
in a system where a primer labeled via an amino-linker is used. The
most important measure to reduce dye interference is proper calibration
of the color compensation feature of the LightCycler software. From our
experience, it is advisable to repeat the color compensation more often
than every 6 months, as is recommended by the manufacturer (17).
This may be dependent on the instrument run
time. However, it cannot be excluded that calibration of the color
compensation per se may give results that lead to insufficient
compensation. Therefore, we recommend that the color compensation be
recalibrated whenever a crossover problem is suspected. The color
compensation is a procedure independent of the PCR run
itself. If suboptimal color compensation is suspected, a new
calibration run can be performed in <30 min, and the calibration can
be applied to the already existent data set.
Our cycling program is the same that we used before (10)(13). This allows the PCR to be carried out in the same run together with factor V, prothrombin G20210A, methylenetetrahydrofolate reductase, and apolipoprotein B-3500 genotyping. This adds to the value of the LightCycler instrument for the detection of common disease-causing mutations. If this PCR is performed with reaction mixtures without dimethyl sulfoxide, the resulting melting points will be ~3 °C higher. Dimethyl sulfoxide, which is present in our reaction mixture, is known to lower the DNA melting point by 0.6 °C for each 1% of dimethyl sulfoxide in the reaction mixture (18).
All mutation detection methods not based on sequencing may give misleading results if there are other base exchanges in the vicinity of the mutation of interest (13). With this hybridization-based method, any other imperfect match under the mutation-compatible probe will produce a mismatch with a typical melting curve. Such mutations are extremely rare, and it is unlikely that a melting curve of such a base exchange would be indistinguishable from that of the wild type (13). All cases found to have unusual melting curves with this approach should, therefore, be further clarified either by sequencing or by use of a wild-type-compatible probe.
To our knowledge, we report here for the first time the simultaneous
detection of two mutations in a single tube by PCR of genomic DNA using
two different reporter dyes with the LightCycler color compensation
feature. This approach is a rapid, convenient, and economic alternative
to other methods described to date for the detection of 1AT
deficiency alleles.
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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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1-antitrypsin and the gene for the S variant. Biochemistry 1984;23:4828-4837.
[Medline]
[Order article via Infotrieve]
1-antitrypsin gene (erratum published in J Biol Chem 1987;262:10412). J Biol Chem 1986;261:15989-15994.
1-Antitrypsin deficiency. Scriver CR Beaudet AL Sly WS Valle D eds. The metabolic and molecular bases of inherited disease 7th ed. 1995:4125-4158 McGraw-Hill New York. .
1-antitrypsin [Technical Brief]. Clin Chem 1999;45:688-690.
1-proteinase inhibitor deficiency alleles Pi*Z and Pi*S by DNA analysis. Eur J Clin Chem Clin Biochem 1996;34:761-764.
[Web of Science][Medline]
[Order article via Infotrieve]
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