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Clinical Chemistry 53: 384-391, 2007. First published January 26, 2007; 10.1373/clinchem.2006.077776
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(Clinical Chemistry. 2007;53:384-391.)
© 2007 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

High-Throughput Microtiter Well-Based Chemiluminometric Genotyping of 15 HBB Gene Mutations in a Dry-Reagent Format

Kyriaki Glynou1,a, Petros Kastanis1,1, Sotiria Boukouvala1,2, Vassilis Tsaoussis1, Penelope C. Ioannou2, Theodore K. Christopoulos3,4, Joanne Traeger-Synodinos5 and Emmanuel Kanavakis5

1 Medicon Hellas S.A., Gerakas, Greece.
2 Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece.
3 Department of Chemistry, University of Patras, Patras, Greece.
4 Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes, Patras, Greece.
5 Department of Medical Genetics, University of Athens, St. Sophia’s Children’s Hospital, Athens, Greece.

aAddress correspondence to this author at: Medicon Hellas S.A., R&D Department, 5-7 Melitona St., Gerakas 15344, Greece. Fax 30-2106-612-666; e-mail glynou{at}mediconsa.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Hemoglobinopathies are the most common inherited diseases worldwide. Various methods for genotyping of hemoglobin, beta (HBB) gene mutations have been reported, but there is need for a high sample-throughput, cost-effective method for simultaneous screening of several mutations. We report a method that combines the high detectability and dynamic range of chemiluminescence with the high allele-discrimination ability of probe extension reactions for simultaneous genotyping of 15 HBB mutations in a high sample-throughput, dry-reagent format.

Methods: We genotyped the HBB mutations IVSI-110G>A, CD39C>T, IVSI-1G>A, IVSI-6T>C, IVSII-745C>G, IVSII-1G>A, FSC6GAG>G-G, –101C>T, FSC5CCT>C–, IVSI-5G>A, FSC8AAG>–G, –87C>G, IVSII-848C>A, term+6C>G, and HbS (cd6GAG>GTG). The method used comprises the following: (a) duplex PCR that produces fragments encompassing all 15 mutations, (b) probe extension reactions in the presence of fluorescein-modified dCTP, using unpurified amplicons, and (c) microtiter well-based assay of extension products with a peroxidase-antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents to simplify the procedure and assigned the genotype by the signal ratio of the normal-to-mutant–specific probe.

Results: We standardized the method by analyzing 60 samples with known genotypes and then validated by blindly genotyping 115 samples with 45 genotypes. The results were fully concordant with sequencing. The reproducibility (including PCR, probe extension reaction, and chemiluminometric assay) was studied for 20 days, and the CVs were 11%–19%.

Conclusions: This method is accurate, reproducible, and cost-effective in terms of equipment and reagents. The application of the method is simple, rapid, and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ß-thalassemia and sickle cell syndromes, the most common group of severe monogenic disorders worldwide (1), are caused by mutations (usually substitutions, deletions, or insertions of up to a few nucleotides) in the hemoglobin, beta (HBB),3 gene located in the short arm of chromosome 11 (11p15.5). This gene is relatively small (<2000 bp), and although >180 causative mutations have been reported for ß-thalassemia syndromes (http://globin.cse.psu.edu), the spectrum of mutations and their frequency in most populations usually consists of a limited number of common mutations plus a slightly larger number of rare mutations. PCR is an essential step of all current methods used for HBB genotyping. The classic methods (2)(3) include restriction fragment length polymorphism analysis (4), dot-blot and reverse dot-blot with allele-specific oligonucleotide probes (5)(6), amplification refractory mutation system (7), multiplex amplification refractory mutation system (8), competitive oligo priming (9), denaturing gradient gel electrophoresis (10), and direct DNA sequencing. Although accurate and relatively simple, these methods are not suitable for high-throughput screening of many mutations in a large number of samples. Several emerging technologies potentially offer higher sample-throughput. Denaturing HPLC (DHPLC)4 was applied to the rapid screening of 25 different HBB mutations (11). DHPLC separates heteroduplex and homoduplex molecules by ion-pair chromatography under partially denaturing conditions. The location and the nature of the mutations must be established by sequencing, however. To this end, a multiplex minisequencing method combined with DHPLC analysis of the single-base extended products was developed for the simultaneous genotyping of 5 common Southeast Asian ß-thalassemia mutations (12). The method, however, requires enzymic treatment of the PCR product to remove excess primers and dNTPs and 55 cycles of the minisequencing reaction (linear amplification) to generate a sufficient amount of extension products before DHPLC analysis. Alternatively, the minisequencing reaction includes fluorophore-labeled ddNTPs, and the products are purified and analyzed by capillary electrophoresis (13). A method based on hybridization of the PCR product with allele-specific oligonucleotide probes that are specific for either the normal or the mutant allele and are immobilized in microtiter wells was reported for characterization of the 8 most common Mediterranean ß-thalassemia mutations (14). The microelectronic array technology was also applied to the detection of 3 HBB mutations (15). Biotinylated amplified DNA (after purification and desalting) was denatured and directed electronically to specific predetermined sites on an array, where it bound to immobilized streptavidin. Specific probes labeled with Cy-3 and Cy-5 were hybridized, and the chip was imaged. The genotyping process required 4–6 h (excluding PCR). Real-time fluorometric melting curve analysis, especially after real-time PCR, was also applied to the detection of HBB mutations (16)(17)(18)(19). These methods are based on the fact that a heteroduplex has a lower melting temperature than a homoduplex molecule. Both latter methods, however, require the initial purchase of costly equipment.

To characterize HBB gene mutations, we developed an alternative simple method that fulfils all the requirements of a diagnostic DNA test but is also cost-effective and amenable to automation to facilitate relatively high sample-throughput when screening for many mutations. As a model for the development and validation of the method, we detected 15 of the most common HBB gene mutations found in the populations of the Mediterranean basin. The mutations analyzed include intervening sequence (intron) I, nucleotide 110 (IVSI-110) G->A, codon (CD)39 C->T, IVSI-1 G->A, IVSI-6 T->C, IVSII-745 C->G, IVSII-1 G->A, frameshift at codon 6 (FSC6) GAG->G-G, –101 C->T, FSC5 CCT->C–, IVSI-5 G->A, FSC8 AAG->–G, –87 C->G, IVSII-848 C->A, termination codon nt +6 (term)+6 C->G, and HbS (cd6 GAG->GTG). The method comprises (a) a duplex PCR to amplify 2 fragments spanning all 15 mutations of the HBB gene (b) a probe extension (PE) reaction of the amplified products in the presence of a fluorescein-modified nucleotide, and (c) detection of PE products on streptavidin-coated microtiter wells by use of a horseradish peroxidase (HRP)-labeled antifluorescein antibody in combination with a chemiluminogenic substrate. The use of lyophilized reagents for PCR and PE reactions makes the assay readily adaptable into a reagent set format.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Information pertaining to the equipment, materials, and samples used in this study is provided in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue3).

chemiluminometric genotyping by pe
PCR.
The solution composition and cycling conditions are provided in the online Data Supplement.

PE Reaction.
PE reaction mixture containing 5 µM dATP, 5 µM dTTP, 5 µM dGTP, 3.5 µM dCTP, 1.5 µM fluorescein-OBEA-dCTP (F-dCTP), 1.5 pmol biotinylated probe, and stabilizers (dextran, trehalose, and sucrose may be used as stabilizers at concentrations of 2%–8%) in a total volume of 10 µL was lyophilized overnight. The mixture was stored at 4 °C with desiccant and just before use was reconstituted by the addition of a solution containing DNA polymerase buffer, Vent exo– DNA polymerase, and PCR-amplified product. The final mixture contained 10 mmol/L KCl, 10 mmol/L (NH4)2SO4, 20 mmol/L Tris, pH 8.8, 2 mmol/L MgSO4, 1mL/L Triton X-100, 5 µM dATP, 5 µM dTTP, 5 µM dGTP, 3.5 µM dCTP, 1.5 µM F-dCTP, stabilizers, 1.5 pmol biotinylated probe, 0.5 U Vent exo– DNA polymerase, and 1.5 µL of the PCR product (total volume, 20 µL). Quantification of the PCR product before the PE reaction was not required. The PE reactions were performed in a thermal cycler as follows: 1 cycle at 95 °C for 2 min followed by 4 cycles of denaturation at 95 °C for 20 s, annealing at 68 °C for 10 s, and extension at 72 °C for 10 s. For each mutation, 2 PE reactions were performed, one using a probe complementary to the normal allele and the other with a probe complementary to the mutant allele. Probes were 5'-biotinylated, whereas their 3'-end was complementary to the allelic nucleotide.

chemiluminometric assay of extension products and interpretation of results
After the PE reaction was completed, 35 µL of wash buffer (see the online Data Supplement) was added to each PE product. A 50-µL aliquot of the PE reaction was transferred into streptavidin-coated wells (see the online Data Supplement for preparation of the wells), and the mixture was incubated at ambient temperature under gentle shaking for 30 min. The wells were washed 3 times with 300 µL wash buffer. Then, 50 µL 0.11–0.22 mg/L antifluorescein-HRP diluted in wash buffer was added, and the mixture was allowed to react for 30 min. The wells were washed as described above, and the activity of bound HRP was measured in the microplate luminometer by adding 50 µL chemiluminogenic substrate into each well and incubating under gentle shaking for 3.5 min at ambient temperature.

Genotypes were assigned for each mutation by determining the signal ratio of specific to nonspecific PE reaction products. Signals were accepted if the relative luminescence units (RLU) were >103 but <106. The signal ratio was calculated by dividing the RLU measured for the reaction of the probe (N) complementary to the wild-type allele to the RLU measured for the reaction of the probe (M) complementary to the mutant allele. Samples with N/M >4 were assigned as normal for the specific mutation; if N/M <0.2, the sample was considered mutant; and if 0.5 < N/M < 2, the sample was considered heterozygous for the specific mutation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The relative positions of primers and extension probes used in the proposed assay are presented in Fig. 1 . The principle of the genotyping method is illustrated in Fig. 2 .


Figure 1
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  		Figure 1. Upper panel, schematic presentation of the HBB gene and the relative positions of primers and probes, as well as the mutation sites.

Lower panel, agarose gel (2.5%) electrophoresis of the products of duplex PCR, followed by ethidium bromide staining. Lanes 3, 5, and 7 contained amplicons produced from PCRs using 1, 1.5, and 2.5 U Tth DNA polymerase, respectively. The lengths of PCR products were 670 and 754 bp. Lane 1 contained DNA ladder (1 kbp), whereas lanes 2, 4, and 6 contained negative controls of the PCRs (water instead of genomic DNA).


Figure 2
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  		Figure 2. (A), workflow of the genotyping procedure.

After DNA extraction, the sample, DNA polymerase, and its appropriate buffer are used for the reconstitution of the lyophilized PCR mixture. PCR is performed, and the amplicons are transferred in a PCR plate containing the PE reaction mixture in lyophilized form. Finally, PE products are transferred in a streptavidin-coated microtiter plate for the chemiluminometric detection. (B), principle of PE reactions and the chemiluminometric detection of PE products. For each mutation 2 biotinylated probes are extended in 2 separate reactions. The 3'-end of the probes is complementary to the allelic nucleotide, and only the probe that is perfectly hybridized to the PCR product will be extended by DNA polymerase leading to the incorporation of F-dCTP. Biotinylated PE products are captured onto streptavidin-coated wells and HRP-labeled antifluorescein antibody (anti-F-HRP) is added. Anti-F-HRP binds only to the extended product. The activity of bound HRP is measured by adding a chemiluminogenic substrate.

pcr
Duplex PCR produces 2 fragments of the HBB gene, flanking all 15 mutations of interest. The region amplified by primers HBB-FF and HBB-IR (a 754-bp fragment, lying between the 139th nucleotide before the cap site at exon 1 and the 140th nucleotide at intron IVSII) contains 12 loci of interest, and the 2nd fragment, amplified by primers HBB-IF and HBB-FR (670 bp, lying between the 258th nucleotide before the exon 3 and the 149th nucleotide after exon 3), flanks 3 loci.

PCR conditions and concentrations of the components were optimized for each of the 2 products individually, and then the optimized conditions were combined in a single reaction. The optimum concentration of each primer needed to produce amplicons of comparable concentrations in the duplex PCR was 250 nM, and the optimum amount of Tth polymerase (not hot start) was 2.5 U. Any thermostable DNA polymerase could be used instead of Tth polymerase, provided that the reaction conditions are optimized accordingly. Proofreading polymerases are not suitable for this assay because the protocol does not include a purification step before PE reaction. Thus, if the proofreading polymerase has retained any activity, it will affect the extension of the probes by editing any mismatches formed between the probe and the template of the reaction. Under the optimized conditions 2 specific products of comparable intensity are generated (Fig. 1Up , lower panel).

pe reaction
The method described here was developed to detect 14 ß-thalassemia mutations and HbS in a single run. All probes used for the PE reactions were 5'-biotinylated to enable capture of PE products onto streptavidin-coated wells, and their 3' end was complementary to the polymorphic nucleotide. To increase the specificity of the reactions, some of the corresponding probes were redesigned to have a mismatch at the 3rd base from the 3' end. The mismatches are highlighted with boldface letters in Table 1 in the online Data Supplement.

PE reaction conditions were optimized for several reasons: (a) to achieve incorporation of F-dCTP into extended products, (b) to avoid nonspecific PE, and (c) to detect all 15 mutations in a single run under the same conditions. For each mutation, optimizations at various conditions were performed with plasmid or genomic DNA samples with known genotypes. The detection of the CD39 mutation required the most stringent conditions to differentiate between normal and heterozygous samples, so the conditions selected for this mutation were subsequently applied to the differentiation of normal, mutant, and heterozygous samples for all the other mutations. In the case of rare mutations, such as FSC6, IVSII-1, –87, FSC5, IVSI-5, FSC8, IVSII-848, term+6, and –101, for which no homozygous mutant samples were available, plasmid constructs were used as templates (106 copies of each plasmid), providing amplicons of comparable concentration with those generated by the amplification of genomic DNA. This procedure ensured similar concentrations in all cases for templates for the PE.

Compared with the modified dUTPs, which are commonly used in PE reactions, the modified dCTPs are not well incorporated by DNA polymerases. Thus, we tested Tth, Klenow exo–, and Vent exo– DNA polymerases, which lack 3'->5' exonuclease activity, for their ability to introduce F-dCTP into the extended products. Vent exo– DNA polymerase gave the best results and was selected for the PE reaction.

The effect of the activity of Vent exo– DNA polymerase was studied in the range 0.2–0.75 U. A normal sample and a CD39 heterozygous sample were analyzed in parallel, N/M was determined at each enzyme activity, and then the ratio N/M (normal)/N/M (heterozygous) was calculated and plotted vs the enzyme activity. The results are presented in Fig. 3 . The ratio N/M (normal)/N/M (heterozygous) increased with increasing enzyme activity up to 0.5 U and reached a plateau at 0.5 U, which was chosen for subsequent studies.


Figure 3
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  		Figure 3. Effect of activity of Vent exo– DNA polymerase on the discrimination between normal and heterozygous CD39 samples.

Both samples were analyzed in parallel with PE reactions using various amounts of Vent exo– DNA polymerase. The ratio N/M (normal)/N/M (heterozygous) was plotted vs the enzyme activity.

The annealing temperatures of the probes to the PCR product, as well as the extension temperature for the PE reaction, were studied in the range of 65–74.5 °C to select those temperatures at which the extension of the noncomplementary probe would be insignificant. The annealing temperature of 68 °C gave the highest N/M ratios for a normal sample. The extension temperature did not affect the N/M ratio, so 72 °C was chosen. The annealing and extension times were studied in the range 0–20 s. It has been reported that reduced extension times increase the discrimination between normal and heterozygous samples (20). The highest ratios for a normal sample were achieved with annealing and extension time of 10 s.

patient study
The method was developed for the detection of 15 ß-thalassemia mutations common in the populations located around the Mediterranean. Initial experiments to optimize the method were performed using 60 samples with known genotypes. Once all the conditions were optimized, the method was validated by blindly analyzing 118 samples with 45 different genotypes. The various genotypes tested and the number of samples corresponding to each genotype are presented in Table 1 . The genotype in 3 samples could not be assigned, because all PE reactions for all mutations generated signals <1000 RLU for each sample (after 2 attempts), probably attributable to poor DNA quality or quantity. For 3 other samples the analysis had to be repeated; on the first analysis the signal N/M ratios were outside the 3 categories for assigning the genotype, but this problem was corrected upon sample reanalysis. The distribution of samples according to the calculated ratios for the mutation IVSI-1 is presented in Fig. 4 (for the distribution of ratios for the rest of the mutations, see Fig. 1 in the online Data Supplement).


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  		Table 1. Patient genotypes analyzed in a blinded study.


Figure 4
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  		Figure 4. Genotyping of samples according to the N/M ratios for the mutation IVSI-1.

N/M ratio is plotted vs the luminescence signal of normal primer. N/M ratios were between 0.5 and 2 for heterozygous samples, increased to higher than 4 in normal samples, and decreased to lower than 0.2 in homozygous mutant samples.

Samples carrying mutations in the same nucleotides as the assay or adjacent to those included in the assay were analyzed to check for false-positive results. The IVSI-5 G>T mutation did not give extension products with probes used for IVSI-5 G>A. Also, samples with either the FSC6 or the HbS mutation gave no extension products with probes used for HbS or FSC6, respectively. In the case of samples with mutant FSC6 or HbS alleles, neither the N nor the M probe for HbS or FSC6, respectively, hybridized perfectly to the amplicon, and neither was extended, resulting in low and approximately equal signals at the chemiluminescent assay for both the normal and mutant reactions. Thus, samples homozygous mutant for FSC6 (or S) gave homozygous genotype for FSC6 (or S), but also heterozygous genotypes for S (or FSC6). The correct interpretation of the test results in this case was that the sample was homozygous for FSC6 (or S) and not heterozygous for S (or FSC6). Good laboratory practice, however, recommends that genotyping results in samples with thalassemia or hemoglobinopathies should always be evaluated and assigned in the light of hematological and clinical findings (21).

Moreover, the method could not differentiate between FSC8.9 and FSC8. Samples carrying the FSC8.9 mutation were detected as FSC8.

reproducibility study
To determine the overall reproducibility (including PCR, PE reaction, and chemiluminometric assay), DNA from 15 heterozygous samples, each carrying 1 of the mutations of interest, was analyzed by the entire protocol on 20 consecutive days. CVs of N/M ratios ranged from 11.1% to 19.0%. The %CV corresponding to each mutation is presented in Table 2 .


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  		Table 2. Reproducibility of the method for the analysis of heterozygous samples of each mutation.1


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Diagnosis of individuals with thalassemia and/or associated hemoglobin structural variants (hemoglobinopathies) is essentially based on hematological, biochemical, and, when appropriate, clinical and family studies. These findings may provide essential clues to the different interactions present in a patient or whether a carrier has thalassemia or a hemoglobin variant, but they do not provide a definitive diagnosis. Definitive diagnosis, through the molecular analysis of underlying genotype(s), is required to direct appropriate management (patients) and support counseling (patients and/or carrier couples) and is fundamental for prevention (carrier couples), including the option of prenatal diagnosis.

Specificity, rapidity, simplicity, and cost are among the most important considerations when selecting a method for routine use in a molecular diagnostics laboratory. The principle of the proposed method is illustrated in Fig. 2Up . The method involves the following steps (Fig. 2Up ): (a) duplex PCR to amplify the genomic region of interest, (b) PE reaction of the amplified product in the presence of F-dCTP and a DNA polymerase lacking 3'->5' exonuclease activity, and (c) immobilization of the PE products in streptavidin-coated wells and chemiluminometric detection of the bound DNA. High specificity of the present method was achieved by optimizing the conditions of each step of the procedure.

Duplex PCR was performed with unlabeled primers, for which the conditions were optimized to achieve the best yield and high specificity of the primers (Fig. 1Up ). It should be noted that duplex "simple" PCR is much easier to optimize than duplex (multiplex) allele-specific PCR, for example amplification refraction mutation system PCR, which requires highly stringent conditions to prevent accumulation of nonspecific products (22). The allele-discrimination step was based on allele-specific PE rather than hybridization with allele-specific oligonucleotides, because it has been reported that the former provides a 10-fold higher discrimination between alleles, due to the selectivity of the DNA polymerases (23). Furthermore, in the proposed method there was no need for a time-consuming amplicon purification step, usually needed for most PE methods (24)(25), because the high detectability of the chemiluminometric assay allows a 50–100-fold dilution of the PCR products before the extension reaction. Primers and nucleotides are also diluted to concentrations that preclude any possible interference with the incorporation of the labeled nucleotide. Another consequence of the high detectability offered by chemiluminescence is that the optimized PE protocol used in this method required only a few cycles, achieving high allele specificity with an absence of nonspecific PE. However, not all polymerases tested were able to introduce the modified F-dCTP nucleotide into the extended product, especially with only a few cycles of extension. We found that this was possible with the use of Vent exo– DNA polymerase. PE reactions using other DNA polymerases required higher numbers of cycles to incorporate F-dCTP.

Because of low background signals, chemiluminometric detection has the benefit of a broad dynamic range, leading to better assay performance than photometric and fluorometric methods and clear discrimination between signals derived from extension of the probes complementary to each allele. The validation of the protocol was clearly demonstrated by the study of patient samples. Bioluminometric methods are gradually being introduced to SNP detection protocols. For example, firefly luciferase was used for the bioluminometric assay of pyrophosphate release in PE reaction, but the method has not been applied to the detection of the HBB gene mutations (26)(27). The photoprotein aequorin was used as a bioluminescent reporter for the detection of only 1 mutation of the HBB gene using the oligonucleotide ligation reaction (28).

Lyophilization of the PCR and PE reagent mixtures and drying of streptavidin-coated wells were advantageous for 2 reasons. First, as demonstrated by accelerated stability tests, the shelf-life of lyophilized and dry reagents is longer than 12 months when stored at 4 °C. All reagents were found to be stable at 37 °C for more than 11 days, and it is well established that stability of reagents for 7 days at 37 °C is equivalent to 1 year at 4 °C (29). Second, the use of preprepared lyophilized and dried reagents in a microtiter plate format makes the entire protocol much faster: 2 h for PCR, 15 min for PE, and 65 min for detection [i.e., total time to complete the genotyping is ~3.5 h (1.5 h after PCR)].

The results of the blind study of patient DNA (115 samples) were fully concordant with the previously characterized genotypes. The variation of the RLU readings for different mutations was attributed to the differences in the hybridization efficiencies between probes and amplicons during the PE reaction and the number of incorporated labeled nucleotides per reaction. The variation of signals between samples analyzed for the same mutation was probably due to the different concentrations of PCR products. However, this variation in the signal does not interfere with the correct assignment of the genotype.

In conclusion, we have developed a robust and high-throughput method for genotyping 15 ß-thalassemia mutations in a simple format that has significant advantages over other, similar methods. A diagnostic reagent set based on this method has been manufactured, which provides the lyophilized and dry reagents ready to use, making the application of the whole procedure simple and rapid. The handling operations by the final user are minimized and, as a consequence, the risk of contamination is greatly reduced. However, different areas for pre-PCR and post-PCR procedures should be used. As an additional precaution uracil glycosylase may be used. Validation of the reagent set with genomic DNA samples demonstrated that it is highly accurate and reproducible. Finally, the method is cost-effective, in terms of equipment (microtiter plate luminometer) and reagents. A standard microplate luminometer costs ~$15 000–$20 000. The cost of reagents and consumables for the analysis of 15 mutations was ~$25 per sample. We estimate that 12 samples can be analyzed (each for 15 mutations) by 1 person in a working day. Also, the method is easily amenable to automation, because there are commercially available pipetting stations that facilitate automation of microtiter well-based assays. Despite the explosive development of microarray technology, the microtiter well-based assay format remains the best approach when a large number of samples are interrogated in parallel for several mutations (high sample-throughput). Although the present method has been developed for the analysis of HBB mutations responsible for ß-thalassemia, it might also be applied to other genes.


   Acknowledgments
 
We thank Gerasimos Dimissianos for assisting in some of the clinical validation experiments.


   Footnotes
 
1 Current address: Directory of Rural Development, Trifilia, Messinia, Greece.

2 Current address: Department of Molecular Biology and Genetics, Democritus University of Thrace, Alexandroupolis, Greece.

3 Human gene: HBB, hemoglobin, beta.

4 Nonstandard abbreviations: DHPLC, denaturing HPLC; PE, probe extension; HRP, horseradish peroxidase; F-dCTP, fluorescein-OBEA-dCTP; RLU, relative luminescence unit(s).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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