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Articles |
University of Turku and Turku University Hospital, Departments of
1
Clinical Chemistry and
2
Medicine, FIN-20520 Turku, Finland.
a Author for correspondence. Fax 358-2-2613920; e-mail veli.kairisto{at}utu.fi
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Abstract |
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Methods: We combined three previously described principles: allele-specific PCR, mutagenically separated PCR, and amplicon identification by specific dissociation curves. PCR amplification was performed with fluorescence detection or conventional thermocycler using the same primers, reactant constituents, and cycling protocol. Primer cross-reactions were prevented by deliberate primer:primer and primer:template mismatches.
Results: PCR products were identified by their characteristic melting temperatures based on SYBR Green I fluorescence. For each of the 256 random and 17 known HFE C282Y samples, mutant homozygous, wild-type, and heterozygous samples were unequivocally distinguished.
Conclusions: This homogeneous assay is rapid, reproducible, does not require fluorescent oligonucleotide probes, and correctly identifies HFE genotypes.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 in 1000 persons (1). Between 64% and
100% of HH patients (depending on the population studied) are
homozygous for a one-base difference at cDNA position 845 of the
hemochromatosis gene (HFE) (2)(3)(4)(5)(6)(7). This
G-to-A transition produces a cysteine-to-tyrosine change at amino acid
282 (C282Y) of the HFE protein (2). A considerable
proportion of HH patients are wild-type/wild-type at the C282Y
HFE locus, indicating that additional genetic defects may be
responsible for this disease (8). A second HFE
mutation occurs at nucleotide position 187 (C-to-G) and changes amino
acid 63 from histidine to aspartic acid (H63D). H63D may contribute to
increased hepatic iron concentrations but does not produce iron
overload in the absence of C282Y. Recently, a new HFE polymorphism,
S65C, which leads to a serine-to-cysteine substitution in exon 2 was
identified (1). This variant is enriched in HH patients, but
whether it contributes to iron overload in HH patients remains
controversial (9). The ability to carry out rapid DNA analysis to determine the mutational status or genotype of an individual has become an increasingly important task for the clinical diagnostic laboratory. Consequently, there is a need for high-throughput and automatable assays to identify clinically significant, single nucleotide polymorphisms (SNPs). Currently, a wide variety of methods exist for detecting single base changes in a DNA molecule. Classical techniques include restriction isotyping (10), single-strand conformation polymorphism analysis (11), oligonucleotide ligation assay (2), heteroduplex analysis (12), and allele-specific (AS) oligonucleotide hybridization probes (3). A simple and inexpensive way to determine the genetic status of an individual is by the use of AS-PCR. In this method, an oligonucleotide primer is specially designed to match one allele but mismatch the other allele at or near the 3' end. If the DNA polymerase cannot extend a primer with a 3' mismatch, one allele is preferentially amplified over the other. The specificity of the AS primers can be further enhanced by engineering a deliberate base change very close to their 3' ends.
To identify a bi-allelic polymorphism, two physically separate PCR reactions are required for each analysis. In addition, a pair of control primers that amplifies an independent fragment usually is included in the reaction to ensure that the PCR reaction itself was successful (13). This method is known by a variety of names, allele-specific amplification, amplification refractory mutation system, and PCR amplification of specific alleles (14). Several methods based on this principle have been developed to detect the C282Y mutation in the HFE gene (15)(16)(17)(18)(19)(20)(21).
An enhanced approach known as PCR amplification of multiple specific alleles (22), or mutagenically separated PCR (23), allows both AS oligonucleotides to be coamplified and differentiated in a single PCR reaction. Cross-reactions between the different AS primers are avoided by the use of deliberate mismatches at or near the 3' and 5' ends of the primers. Compared with AS amplification, mutagenically separated PCR eliminates the need for an internal control primer set and reduces the cost and labor of the techniques by approximately one-half. Merryweather-Clarke et al. (24) have applied this method to the detection of the HFE C282Y genotype. However, this technique and the aforementioned methods are not ideally suited to large-scale analysis because they require a laborious post-PCR processing step.
The problems of low throughput and the requirement for postamplification manipulations have been overcome by the development of new PCR instruments that can monitor the PCR reaction in real time. These devices are composed of a thermal cycler coupled to a fluorescent detector and are capable of PCR amplification with simultaneous amplicon analysis (25)(26). A simple approach for concurrent DNA amplification and detection is the use of AS primers and a fluorescent double-stranded DNA-specific binding dye (SYBR Green I). Products are detected by their characteristic dissociation profiles. A product dissociation profile is generated after the PCR reaction by monitoring the fluorescence of the SYBR Green I dye as the temperature passes through the amplicon’s denaturation temperature. Dissociation profiles are dependent on the GC content and length and sequence of the PCR products (25).
Germer and Higuchi (27) recently applied this strategy to identify SNPs of the paraoxonase and apolipoprotein B genes. In a method the authors call Tm-shift genotyping, a 5' GC-tail of 26 base pairs is attached to one of the AS primers. This functions by increasing the melting profile of that primer and thus allows it to be differentiated from the second AS primer.
We now report a real-time, single-tube, homogeneous SNP detection method that combines the principles of Newton et al. (13), Rust et al. (23), and Germer and Higuchi (27), and we show its applicability to the rapid detection of the three different HFE C282Y genotypes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dna extraction
Genomic DNA was extracted from 3 mL of EDTA-anticoagulated blood.
The DNA isolation was carried out using a salting-out method
(28). Blood cells were lysed with 7.5 mL of Tris buffer 1,
pH 8.0 (13 mmol/L Tris base, 10 mmol/L KCl, 14.5 mmol/L
MgCl2 · 6 H2O, 2 mmol/L
EDTA, and 25 mL/L Triton X-100). After centrifugation (7 min at
2400g), the pellet was washed with Tris buffer 1 and
centrifuged (7 min at 1200g). The pellet was then lysed with
660 µL of Tris buffer 2, pH 8.0 (13 mmol/L Tris base, 10 mmol/L KCl,
4.7 mmol/L MgCl2 · 6
H2O, 2 mmol/L EDTA, 0.4 mol/L NaCl, and 10 g/L
sodium dodecyl sulfate), and incubated for 15 min at 56 °C. Cellular
proteins were removed by precipitation with 300 µL of 5 mol/L
NaCl and centrifugation (7 min at 560g). DNA was
isolated by ethanol precipitation and incubated for 1 h at 4 °C
in Tris-EDTA buffer, pH 8.0 (13 mmol/L Tris base, 1 mmol/L EDTA). The
DNA concentration was then measured spectrophotometrically at 260 nm,
and samples were diluted to a final concentration of 20 mg/L (20
ng/µL).
pcr primers
Three oligonucleotide primers were designed based on the National
Center for Biotechnology Information GenBank HFE cDNA
sequence (Accession No. U91328). A schematic representation of the
different oligonucleotide primers used for genotyping the C282Y locus
is shown in Fig. 1
. The wild-type, mutant, and common primers (sequences) were
HFEW2 (5'-GGG GGG CCC CGG GCC CAG ATC ACA ATG AGG GGC ACA TCC AGG CCT
GGG TGC TCC ACC TCG C-3'), HFEM (5'-TGA TCC AGG CCT GGG TGC TCC ACC TGC
T-3'), and HFECOM (5'-CAG GGC TGG ATA ACC TTG GCT GTA CC-3'),
respectively. The primers were purchased from MedProbe.
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To allow product identification from the single reaction mixture, the AS primers were designed with different lengths according to Rust et al. (23). The two forward AS primers, HFEW2 and HFEM, were 61 and 28 bp long, respectively, and the complementary primer, HFECOM, was 26 bp in length. Mispriming and cross-reactions were prevented by the introduction of deliberate mismatches between primers and template (13)(23).
The first nucleotide difference (C or T) between AS primers HFEW2 and HFEM is located at the 3' terminal base. To ensure the specificity of these primers, a DNA polymerase that lacks the 3' exonuclease proofreading activity (DyNAzyme II) was used in the PCR reaction. The second primer base change (G to C) generates a purine/pyrimidine primer/template mismatch, and this prevents amplification of the nonmatching AS primer. This mismatch is located three bases from the 3' end of HFEW2 and two bases from the 3' end of HFEM. Two additional nucleotide changes (A and C) were made to the HFEW2 primer, located at the same positions as the last two 5' nucleotides of the HFEM primer. These changes should prevent the generation of possible spurious products, which could otherwise occur by the annealing and extension of the HFEM primer to the first-round product of HFEW2. Finally, to facilitate the discrimination of the AS primers by dissociation curve analysis, a 13-bp GC tail was added to the 5' end of the HFEW2 primer.
pcr amplification
PCR reactions were carried out using MicroAmp Optical
TubesTM and MicroAmp Optical
CapsTM (PE Biosystems). The PCR reaction mixture
contained the following in a final volume of 25 µL: 50 ng of genomic
DNA, PCR reaction buffer (10 mmol/L Tris-HCl, pH 8.8, 1.5 mmol/L
MgCl2, 50 mmol/L KCl, and 1 mL/L Triton X-100;
Finnzymes), 5 mmol/L dNTP, 1 U of DyNAzyme II DNA Polymerase
(Finnzymes), 5 pmol of HFECOM and HFEW2, 20 pmol of HFEM, and 2.5 µL
of SYBR Green I 1:10 000 (Molecular Probes). Negative control
reactions containing water in place of DNA were included in each batch
of PCR reactions to ensure that contamination was not a problem. The
PCR amplification profile was as follows: initial denaturation at
95 °C for 4 min, followed by 32 cycles of denaturation at 96 °C
for 30 s and combined annealing and extension at 71 °C for
30 s. To investigate the versatility of the method, we carried out
PCR amplification in two different thermocyclers, the PTC-200 DNA
engine (MJ Research) and the GeneAmp® 5700
Sequence Detection System (PE Biosystems)
product analysis
In the GeneAmp 5700, analysis of the real-time
fluorescence signal from SYBR Green I bound to double-stranded DNA was
performed by the GeneAmp 5700 software (PE Biosystems). A threshold
cycle (Ct) was determined for each sample, using
the exponential growth phase and baseline data of the fluorescent
amplification plots. A sample was deemed positive if it had an increase
in fluorescence above the Ct. The fluorescent
amplification plots were converted to dissociation curves by plotting
the negative derivative of fluorescence with respect to temperature.
Dissociation curves were subsequently used to identify PCR products.
For confirmatory electrophoretic analysis of PCR products, a 10-µL
aliquot of each reaction was run on a 2% gel in Tris-borate-EDTA
buffer.
dna sequencing
PCR products for sequencing were generated using the primer
HFESEKV (5'-TTA CCT CCT CAG GCA CTC CTC TCA ACC-3') and HFECOM primers.
Twelve different samples were analyzed by automated "Dye
Terminator" cycle sequencing using an ABI 377 automatic sequencer (PE
Biosystems) with DNA cycle sequencing reagent set (BigDye Terminator
Cycle Sequencing FS Ready Reaction Kit; PE Biosystems) according to the
manufacturer’s protocol.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This was a highly effective way to quickly optimize the assay
in contrast to endpoint analysis, which yielded considerable
fluorescence variation, especially before the number of PCR cycles was
optimized and reduced to 32 (data not shown).
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identification of wild-type, heterozygous, and homozygous samples
The results for the HFEW2, HFEM, and HFECOM primers with the
GeneAmp 5700 Sequence Detection system are shown in Fig. 3
A. For each C282Y sample, the AS primers accurately
distinguished between mutant homozygote, wild type, and heterozygote.
The melting of the sample homozygous for the 845 G allele showed a
marked change (decrease) in fluorescence between 86 and 88 °C, with
a maximum rate of change around 87 °C. In contrast, the sample
homozygous for the 845 A allele showed a marked decrease in
fluorescence between 82 and 84 °C, with a maximum rate of change
around 83 °C. The heterozygous sample contained both fluorescent
melting peaks because of the presence of amplicons derived from both
alleles. Confirmatory analysis of the products by slab gel
electrophoresis revealed that the wild-type samples generated the
expected 113 bp with the HFEW2 primer and no product was amplified with
the HFEM primer. Similarly, the HFEW primer generated no product with
the mutant homozygous sample, but as expected, the HFEM primer
generated a band of 80 bp. For the heterozygous sample, both the 80-
and 113-bp products were visible on the gel.
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We next tested the possibility of using a standard thermocycler
(PTC-200 DNA Engine; MJ Research) to amplify the C282Y locus. We then
used a short 20-min dissociation protocol on the 5700 machine to
analyze the PTC-200 products; the results are presented in Fig. 3B
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These results show that the dissociation curves produced by the
products of either thermocycler are nearly identical.
assay performance
To test the reproducibility of the fluorescence dissociation
curves, 10 wild-type, 10 heterozygous, and 5 mutant homozygous samples
were analyzed in duplicate (Fig. 4
). For each allele, the dissociation curve was highly
reproducible: the sample-to-sample and within-sample variations of the
dissociation curves were <0.5 °C. The robustness of the technique
was evaluated by analyzing >200 DNA samples, and all samples tested
gave an unambiguous C282Y HFE genotype.
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assay validation
The validity of the method was verified independently by testing
17 samples comprising all three C282Y HFE genotypes. Twelve samples (4
mutant homozygous, 4 heterozygous, and 4 wild-type homozygous) were
sequenced using HFESEKV. Five other samples (one mutant homozygous, two
heterozygous, and two wild-type homozygous) were analyzed by an outside
reference laboratory using a restriction digestion method
(29). The genotype results obtained for all 17 samples were
100% concordant to those obtained by the current method.
genotype scoring
The SDS 5700 software allows the export of numeric dissociation
curve data to other software. We exported the data into Microsoft Excel
and designed a macro program that calculated the area under the
dissociation curve. Subsequently, a scatter graph was generated in
which the area under the dissociation curve between temperatures 81.8
and 83.6 °C was plotted on the x axis and the area under
the dissociation curve between temperatures 85.8 and 87.3 °C was
plotted on the y axis. This generated a graph in which the
three C282Y genotypes and the no-template controls separated into four
discrete clusters with definable limits (Fig. 5
). Using this customized Excel sheet, we were able to automate
the process of genotype scoring. Of the 256 random-study subjects,
there was 1 mutant homozygous and 19 heterozygous individuals. Thus,
the overall allele frequency for HFE Tyr 282 was 4.1%, and the carrier
frequency was 7.8%.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many individuals with hemochromatosis (>60%) are homozygous for the
missense mutation C282Y. Compound heterozygosity or homozygosity for
the H63D mutation are also associated with hemochromatosis, but with
very low penetrance. No more than 1% of the compound
heterozygotes will develop hemochromatosis. This value is even lower
for H63D homozygotes. Thus, 99% of compound heterozygotes that
would be found in either a subpopulation of relatives of C282Y
homozygotes or in the general population would be false positives
(1). Therefore, we believe that H63D genotyping is relevant
only for C282Y heterozygotes and only in those cases where clinical
suspicion of hemochromatosis remains, as assessed by biochemical tests
such as plasma ferritin and transferrin saturation. For a clinical
laboratory engaged in hemochromatosis screening, the first-line genetic
test is C282Y genotyping.
In this study, we have demonstrated the feasibility of using real-time
AS-PCR and DNA dissociation curves to genotype the C282Y HFE
locus. Compared with the widely used C282Y restriction isotyping assay,
our method is less time-consuming and labor-intensive because it does
not require any post-PCR processing. In addition, reports by both
Jeffrey et al. (32) and Somerville et al. (33)
have shown that the restriction isotyping assay, when used with the
primers of Feder et al. (2), may have the potential to
incorrectly classify a C282Y heterozygote as a C282Y homozygote. This
is caused by a newly identified SNP (5569 G/A) located in the binding
region of the Feder et al. (2) antisense primer. The primers
used in our method avoid this polymorphism (Fig. 1
).
Recently, several real-time HFE PCR genotyping methods based
on the principle of fluorescence resonance energy transfer have been
developed (34)(35)(36). These methods use fluorescently labeled
oligonucleotide probes. In contrast, our real-time C282Y assay uses
novel AS primers and the inexpensive SYBR Green I DNA-binding dye to
generate AS dissociation curves. Our report demonstrates that this
approach is both reliable and robust. This is illustrated by the high
degree of reproducibility obtained for each different allele
dissociation curve. These curves were generated using several different
individuals (Fig. 4
). Previously, we have shown that DyNAzyme II, a
Thermus brockianus enzyme, functions specifically in
multiplex AS-PCR (37). This observation is confirmed by the
present report.
The capital costs of the GeneAmp 5700 Sequence Detection System are
high. Therefore, to maximize productivity, the machine should be used
for a variety of different assays. Consequently, machine time available
to individual users may become scarce. We have shown, however, that it
is possible to perform the PCR step in a conventional thermocycler and
that a subsequent 20-min dissociation curve analysis of the products by
the GeneAmp 5700 system gives unequivocal results (Fig. 3B
). Thus, an
added benefit of this genotyping technique is its versatility, which
yields substantial savings in machine time.
A potential disadvantage of the current method is that SYBR Green I
binds to all amplification products, including primer dimers, and this
could cause difficulty in identifying the intended amplicon. Although
primer dimer formation did occur during the initial optimization phase,
the primer dimers were differentiated from the desired amplicons by
differences in melting temperatures. Moreover, the formation of primer
dimers was prevented by a combination of increased annealing
temperature, alteration in the ratio of primers, and a reduction in the
number of PCR cycles (Fig. 2
). We found that, as reported by Rust et
al. (23), it was necessary to use the longer AS primer
(HFEW2) at a lower concentration than the shorter primer (HFEM). This
probably reflects the greater annealing advantages of longer primers at
high temperatures.
This assay format should represent a straightforward way to genotype the C282Y HFE locus. Theoretically, a similar strategy could be applied to the analysis of other clinically established SNPs. However, as Germer and Higuchi (27) pointed out, it remains to be seen whether all SNPs can be genotyped using specially designed AS primers and SYBR Green I. The fact that some SNP targets are very GC rich may make the optimization and discrimination of their PCR products difficult, but not necessarily impossible. Thus, an obvious aid for designing good PCR primers for use in this system is a method that can accurately predict the melting temperature of the resulting PCR product. We calculated the theoretical melting temperature for the HFE products, using the equation of Howley et al. (38) as described by Bohling et al. (39). Comparison of the theoretical melting temperature with the measured melting temperature revealed an overestimation of 2.4 °C for the HFEM amplicon and 3.7 °C for the HFEW2 amplicon. This discrepancy, as Bohling et al. (39) have suggested, probably results from the addition of SYBR Green I, which has been shown to affect dissociation curves in a concentration-dependent fashion. In addition, the equation developed by Howley et al. (38) itself comes from low-resolution melting experiments in the 1960s and 1970s, and therefore is imprecise. Thus, both of these factors probably account for the discrepancy. Additional work will be needed to create an algorithm for accurate prediction of oligonucleotide melting temperatures in a buffer containing SYBR Green I.
In conclusion, a new single-tube HFE genotyping method that combines the principles of AS-PCR, mutagenically separated PCR, and amplicon identification by SYBR Green I dissociation curves has been developed. The method is accurate and reliable and does not require oligonucleotide probes. It represents a rapid approach for HFE genotype determination.
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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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