| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef Strauss Allee 11, 93042 Regensburg, Germany
a author for correspondence: fax 49 941 944 6202, e-mail Charalampos.Aslanidis{at}klinik.uni-regensburg.de
Apolipoproteins play a central role in cholesterol transport by their association to lipoproteins and their function as ligands for receptors, cofactors, or structural proteins. Mutations and polymorphisms in a variety of apolipoproteins lead to lipoprotein metabolism disorders and/or susceptibility to cardiovascular disease. In familial defective apolipoprotein B-100, the clearance of LDL particles from the circulation is impaired because of reduced affinity of the apolipoprotein (apo) B component of LDL for the LDL receptor as a result of a G-to-A mutation at nucleotide 10708 in exon 26 of the apoB gene, which causes substitution of Arg3500 for Gln (1)(2). The frequency of the mutation is 1 in 700 in the general population (3). Heterozygous individuals have increased serum concentrations of cholesterol. Apolipoprotein E (apoE) likewise is involved in the clearance of HDL, contributing to reverse cholesterol transport (4). Genetic variation at the APOE locus in humans is an important determinant of plasma lipid concentrations and relative risk of atherosclerosis (5). Among several rare variants, three major alleles have been identified in the population: E2, E3, and E4. The most common E3 isoform is distinguished by cysteine at position 112 and arginine at position 158 in the receptor-binding region of apoE. The E4 isoform (Arg112 and Arg158) is associated with increased cholesterol, thus enhancing the risk of heart disease (5). In addition, E4/E4 individuals have a very high risk for developing Alzheimer disease(6). Most patients with type III hyperlipidemia are homozygous for the E2 allele (Cys112 and Cys158).
To date, genotyping for apoE is mainly performed by PCR, followed by digestion with restriction enzymes and restriction fragment length polymorphism (RFLP) analysis, and separation of the resulting DNA fragments on agarose or acrylamide gels (7). For apoB3500, an allele-specific PCR method is widely used (8). These nonhomogeneous methods give rise to unequivocal results, but they are time-consuming and require optimization of the PCR reaction to eliminate nonspecific PCR products, which would disturb the genetic analysis. Recently, a new detection methodology that relies on hybridization of amplicon-specific oligonucleotides with adjacent fluorophores capable of fluorescence resonance energy transfer has been introduced and used in a new high-speed thermal cycler (LightCycler; Roche Diagnostics) that uses glass capillaries and hot air for heating(9). This technology allows the real-time detection of the specific amplicon, followed by detection of the mutation by identification of the melting behavior of one of the two hybridization oligonucleotides (10). To this end, one hybridization primer matches the wild-type sequence (or mutant sequence), with the variable nucleotide 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 proximally by a distance of one to three nucleotides and is labeled with fluorescein at its 3' end. During cycling, the hybridization probes hybridize the specific PCR product at the annealing temperature, and fluorescence is monitored. Cycling conditions are very fast because of temperature adjustments in the glass capillary (high surface-to-volume ratio). Following completion of the PCR, the PCR mixture is denatured and the temperature is lowered to 40 °C to facilitate binding of the hybridization probes, generating maximum fluorescence, and then slowly increased to 80 °C to permit melting of the detection probe, which is monitored by the decline of the fluorescence. This is being performed in the LightCycler itself, which has an integral device that enables denaturation and fluorescence detection. 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 genomic DNA isolated from EDTA blood from individuals who previously had been typed by PCR-RFLP (apoE) and mutation-specific priming (apoB3500). The primers for the apoE PCR were CA1 5'-TTGAAGGCCTACAAATCGGAACTG-3' and CA2 5'-CCGGCTGCCCATCTCCTCCATCCG-3', which produce a 462-bp PCR product from genomic DNA. The primers for the apoB gene were UOL-A 5'-GGAGCAGTTGACCACAAGCTTAGCTTGG-3' and LOL-A 5'-AGAGTTCCAGGGTGGCTTTGCTTGTATG-3', which produced a 352-bp PCR product. The 3'-phosphorylated detection primer for apoE112 was E4-112M (LC-red640-ACATGGAGGACGTGCGCGG-p). The anchor primer E4-112A (CTGCAGGCGGCGCAGGCCCGGCTGGGCGC-fluorescein) was located proximal to the detection primer at a distance of two nucleotides. Likewise, the detection primer for apoE158 was E2-158M (LC-red640-GACCTGCAGAAGCGCCTGGC-p), and the anchor primer was E158A (GCTGCGTAAGCGGCTCCTCCGCGATGCCG-fluorescein). Amplicon and mutation detection for apoB3500 was facilitated by the detection primer B3500M (red640-AGCTTCACTGAAGACCGTGTGCTCTTGGA-p) in combination with the anchor primer B3500A (GGTTCCAGATATCATCAATTTTGGAAGTGCCCTG-fluorescein) at a distance of two nucleotides.
PCR and melting curve determination were performed in 20-µL volumes in glass capillaries (Boehringer Mannheim). For apoE genotyping, the following pipetting scheme was used: 8.2 µL of H2O, 1.8 µL of 25 mmol/L MgCl2, 1 µL each of 5 pmol/µL CA1 and CA2, 1 µL each of E4-112A and E4-112M (for the apoE112 site) or E-158A and E2-158M (for the apoE158 site) from 4 pmol/µL stock solutions, 2 µL of dimethyl sulfoxide, 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 apoB3500 mutation detection, 5.6 µL of H2O, 2.4 µL of 25 mmol/L MgCl2, 2 µL each of 5 pmol/µL UOL-A and LOL-A, 2 µL each of B3500M and B3500A 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. Cycling conditions were identical for both applications, with initial denaturation at 94 °C for 2 min, followed by 40 cycles of denaturation 94 °C for 0 s, annealing at 60 °C for 10 s, and extension at 72 °C for 15 s, with a ramping rate of 20 °C/s. The fluorescence was monitored at the end of each 10-s annealing phase. After the products were amplified, we generated melting curves by denaturing the reaction at 95 °C for 0 s, holding the sample at 40 °C for 5 s, and then slowly heating the sample to 80 °C with a ramp rate of 0.2 °C/s, simultaneously monitoring the decline in fluorescence. We converted melting curves to melting peaks by plotting the negative derivative of the fluorescence with respect to temperature (-dF/dT) against temperature.
The fluorescence profiles generated from selected DNA samples and
negative controls are shown in Fig. 1
. When the apoE112 hybridization probes were used (Fig. 1
, top
left), fluorescence increased constantly in the samples with the DNA
(samples 3 and 4), whereas no fluorescence was detected in the control
sample (H2O). Although no fluorescence was
detectable in DNA sample 2, analysis of the PCR products on agarose
gels revealed the presence of the specific PCR product. Sample 2 does
not produce fluorescence because the detection hybridization probe for
apoE112 is derived from the E4 allele, which leads to a
mismatch in the PCR products (E3/E3), generating
a low melting point (53.8 °C). The PCR cycling, however, has been
performed at an annealing temperature of 60 °C. A reduction in the
annealing temperature increases fluorescence during PCR cycling when
DNA from an E3/E3 individual is used (data not
shown). The melting curves of the same samples are shown in Fig. 1
(top
right). The melting point (Tm) of the
E3/E3 DNA (curve 2) was 53.8 °C, demonstrating
the presence of specific PCR product in the reaction. The
Tm of the E4/E4 DNA (curve 4)
was 61.8 °C because the detection probe matches perfectly. The
E2/E4 DNA (curve 3) showed melting peaks at 53.8
and 61.8 °C, demonstrating heterozygosity at position E112.
|
The fluorescence monitored with the hybridization probes specific for
the apoE158 position is shown in Fig. 1
(middle left). The three DNA
samples of different genotypes produced different fluorescence signals,
although analysis of the PCR products on agarose gels revealed equal
amounts of PCR product (data not shown). The detection probe of the
apoE158 site was derived from the E4 allele and matched the
sequence of the E4 and E3 alleles perfectly,
leading to stable hybridization at the annealing temperature, whereas
base pairing with PCR products from E2/E2
individuals was impaired because of a lower
Tm (56.2 °C). This apparent
variability in signal intensity did not affect the quality of the
melting curves because monitoring of the melting behavior started at
40 °C, the temperature at which mismatched detection probes
contribute equally to signal generation. The
Tm of the E2/E2
sample (curve 4) was 56.2 °C, whereas the perfect match with the
E3/E3 sample produced a
Tm of 64.2 °C (curve 2). The sample
of the heterozygous individual (curve 3) showed a two-phase melting
behavior.
The detection probes used for apoE genotyping in the LightCycler
revealed 8 °C differences in melting temperatures. A summary of the
melting peaks of individual apoE genotypes is given in Table 1
. The combination of the digits 1 and 0, with 1 meaning that the
particular peak is present and 0 reflecting the absence, is unique and
unambiguously determines the apoE genotype. The four individual
Tms (53.8, 56.2, 61.8, and 64.2 °C)
are well separated. Using all hybridization probes simultaneously in
one PCR reaction, we were able to identify individual melting peaks in
some samples, but reliable genotyping was not possible (data not
shown). Therefore, two separate PCR reactions with the respective
hybridization probes are recommended for apoE genotyping. To date, we
have analyzed >100 individuals with various apoE genotypes with the
LightCycler and compared the data with the results obtained with
traditional PCR-RFLP. The genotypes were identical. The positions and
distances of melting peaks were identical in individuals with the same
genotypes.
|
The detection probe of the apoB3500 mutation was derived from the
nonsense DNA strand and matched the wild-type sequence of the gene.
Because of the length and base composition of the probe, the melting
points were higher compared with the apoE probes. Therefore, the
fluorescence signal intensity during PCR reaction was equally high for
the DNA sample with the wild-type sequence (curve 3 in Fig. 1
, bottom
left) and the heterozygous individual (curve 2). The melting peaks were
clearly differentiated (Tm 68 and
71.5 °C). The rather similar melting behavior (compared with apoE)
is attributable to the length and base composition of the probes. Using
the same procedure as for apoE genotyping, we performed apoB3500
mutation analysis on different wild-type samples (different individuals
and different DNA preparations) and samples from a heterozygous
individual. The results were identical to the results obtained with
gel-based technologies. There was no run-to-run variation of the
melting points.
Compared with traditional PCR genotyping and mutation detection, applications can be adapted very easily on the LightCycler. We have established additional mutation detection protocols for methylenetetrahydrofolate reductase and prothrombin (manuscript in preparation). In our experience, the basis for the design of the PCR primers is the same as required for traditional thermal cyclers and protocols. Extensive optimization is not required; in fact, for apoB3500, methylenetetrahydrofolate reductase, and prothrombin, the PCR primers and cycle conditions did not need to be optimized at all. Because the DNA sequence of the apoE gene is GC-rich, PCR conditions and primers had to be optimized. Because of the nature of the generation of the fluorescence signal, contaminating, nonspecific PCR products do not affect the results. The PCR primers for apoE described in this study generated a specific amplicon. With respect to the hybridization probes, a distance of two nucleotides between the anchor primer and the detection primer was ideal for efficient energy transfer and signal generation. The anchor primer (28–30 nucleotides in length) should enable hybridization at higher temperatures compared with the detection primer (~20 nucleotides in length). The mutant nucleotide should be positioned close to the middle of the detection primer. It is also important that the annealing temperature in the PCR reaction is kept below the melting point of the detection primer to allow monitoring of the fluorescence during the cycling.
In our opinion, the LightCycler technology is more accurate than traditional, gel-based assays because it enables the identification of the specific amplicon and uses this amplicon for mutation detection. It is conceivable that in a traditional method that uses PCR-RFLP, contaminating PCR products would scramble the analysis. In an allele-specific amplification (as performed for apoB3500), the quality of the discriminating primers is crucial.
We have established high-speed (30 min) and easy-to perform-genotyping for the apoE alleles and the identification of the apoB3500 mutation, using the LightCycler technology and melting curves. This homogeneous system minimizes PCR contamination concerns related to sample handling and does not require digestion of PCR products with restriction enzymes and/or fragment separation on gels.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  K. E Liyanage, A. J Hooper, J. C Defesche, J. R Burnett, and F. M van Bockxmeer High-resolution melting analysis for detection of familial ligand-defective apolipoprotein B-100 mutations Ann Clin Biochem, March 1, 2008; 45(2): 170 - 176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Grunenfelder, M. Umbehr, A. Plass, L. Bestmann, F. E. Maly, G. Zund, and M. Turina Genetic polymorphisms of apolipoprotein E4 and tumor necrosis factor {beta} as predisposing factors for increased inflammatory cytokines after cardiopulmonary bypass J. Thorac. Cardiovasc. Surg., July 1, 2004; 128(1): 92 - 97. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Dobrowolski, R. A. Banas, J. G. Suzow, M. Berkley, and E. W. Naylor Analysis of Common Mutations in the Galactose-1-Phosphate Uridyl Transferase Gene: New Assays to Increase the Sensitivity and Specificity of Newborn Screening for Galactosemia J. Mol. Diagn., February 1, 2003; 5(1): 42 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Burggraf, S. Kosel, S. Lohmann, R. Beck, and B. Olgemoller Unexplained DNA Melting Behavior in a Genotyping Assay Clin. Chem., January 1, 2002; 48(1): 199 - 201. [Full Text] [PDF]
|
|
|
|
 |
|
 N. Drabe, G. Zund, J. Grunenfelder, M. Sprenger, S. P. Hoerstrup, L. Bestmann, F. E. Maly, and M. Turina Genetic predisposition in patients undergoing cardiopulmonary bypass surgery is associated with an increase of inflammatory cytokines Eur. J. Cardiothorac. Surg., September 1, 2001; 20(3): 609 - 613. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Foy and H. C. Parkes Emerging Homogeneous DNA-based Technologies in the Clinical Laboratory Clin. Chem., June 1, 2001; 47(6): 990 - 1000. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Press Detection of Prevalent Genetic Alterations Predisposing to Hemochromatosis and Other Common Human Diseases Clin. Chem., October 1, 2000; 46(10): 1526 - 1528. [Full Text] [PDF]
|
|
|
|
|
|
F. A.J.T.M. van den Bergh, A. M. van Oeveren-Dybicz, and M. A.M. Bon Rapid Single-Tube Genotyping of the Factor V Leiden and Prothrombin Mutations by Real-Time PCR Using Dual-Color Detection Clin. Chem., August 1, 2000; 46(8): 1191 - 1195. [Full Text] [PDF]
|
|
|
|
|
|
M. A.M. Bon, A. van Oeveren-Dybicz, and F. A.J.T.M van den Bergh Genotyping of HLA-B27 by Real-Time PCR without Hybridization Probes Clin. Chem., July 1, 2000; 46(7): 1000 - 1002. [Full Text] [PDF]
|
|
|
|
|
|
M. Nauck, M. M. Hoffmann, H. Wieland, and W. Marz Evaluation of the Apo E Genotyping Kit on the LightCycler, Clin. Chem., May 1, 2000; 46(5): 722 - 724. [Full Text] [PDF]
|
|
|
|
|
|
P. S. Bernard and C. T. Wittwer Homogeneous Amplification and Variant Detection by Fluorescent Hybridization Probes Clin. Chem., February 1, 2000; 46(2): 147 - 148. [Full Text] [PDF]
|
|
|
|
|
|
C. Aslanidis, M. Nauck, and G. Schmitz High-Speed Detection of the Two Common {alpha}1-Antitrypsin Deficiency Alleles Pi*Z and Pi*S by Real-Time Fluorescence PCR and Melting Curves Clin. Chem., October 1, 1999; 45(10): 1872 - 1875. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
