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Technical Briefs |
1 Biomedical Science Section, School of Nursing, The Hong Kong Polytechnic University, Hong Kong SAR, China
aaddress correspondence to this author at: Room FG511, Biomedical Science Section, School of Nursing, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR, China; fax 852-2364-9663, e-mail hsspyip{at}polyu.edu.hk
ß-Thalassemias are inherited hemoglobin disorders characterized by reduced production of ß-globin chain. The severe forms of ß-thalassemia produce marked anemia starting a few months after birth, and survival relies on regular blood transfusion and the lifelong use of drugs to prevent iron accumulation (1). ß-Thalassemias are among the commonest genetic disorders in the world, and more than 200 mutations have been described to date. However, each at-risk population has a few common mutations (usually four or five) and a variable number of rare alleles (1). Common Southeast Asian ß-thalassemia mutations include -28 A>G, codon (CD) 26 G>A, CD 41/42 -CTTT, CD 71/72 +A, IVS2 + 654 C>T, and others. The mutation CD 26 G>A produces hemoglobin E, presents like thalassemia, and is particularly common in Thailand, Cambodia, Vietnam, and Malaysia (1).
There are many methods for genotyping known ß-thalassemia mutations (2). Here we describe a rapid method for simultaneous genotyping of these five common mutations. The study was approved by the Human Subject Ethics Subcommittee of the Hong Kong Polytechnic University. The method is based on multiplex minisequencing followed by analysis of the single-base-extended products by denaturing HPLC (DHPLC). In minisequencing, a primer anneals to a DNA template immediately upstream of a mutation site to be analyzed and is extended by a single base as a result of incorporating a dideoxyribonucleotide triphosphate (ddNTP) (3). The single-stranded extension products are then separated by ion-pair reversed-phase liquid chromatography under completely denaturing condition, and the retention time is a function of both size and base composition (4).
The DNA template for minisequencing was first generated by amplifying the whole human ß-globin gene using primers HBBpF1 and HBBpR1 (Table 1
). The 25-µL reaction mixture contained 0.1 µM each of the primers, 50 ng of genomic DNA, 2.0 mM MgCl2, 0.2 mM each of the deoxyribonucleotide triphosphates, and 0.5 U of FastStart DNA polymerase (Roche Applied Science) in 1x PCR buffer [50 mM Tris-HCl, 10 mM KCl, 50 mM (NH4)2SO4, pH 8.3 at 25 °C]. Hot-start amplification was performed in a GeneAmp PCR system 9700 (Applied Biosystems) with initial denaturation at 95 °C for 5 min followed by 35 cycles of 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 2 min, with a final extension at 72 °C for 7 min. The excess primers and deoxyribonucleotide triphosphates were removed by treating 5 µL of PCR product with 5 U of exonuclease I (New Biolabs) and 0.5 U of shrimp alkaline phosphatase (Amersham Biosciences) at 37 °C for 15 min; the enzymes were then inactivated at 80 °C for 20 min.
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Primers (Table 1
) for minisequencing were designed so that they fulfilled the following criteria: (a) the formation of hairpin structure and primer-dimers was minimal; (b) the annealing temperatures were similar for all five primers (excluding the 5' noncomplementary tail); and (c) the primers and their extension products did not overlap during chromatographic elution. The multiplex minisequencing reaction was carried out in a volume of 25 µL containing 5 µL of treated PCR product, 50 µM each of the ddNTPs, 0.5 U of Thermo Sequenase DNA polymerase (Amersham Biosciences), 0.6 µM the primer HBB3Um2, and 0.4 µM each of the other four primers in a 1x reaction buffer (26 mM Tris-HCl, 6.5 mM MgCl2, pH 9.5). Thermal cycling was started with an initial denaturation of 96 °C for 10 s, followed by 55 cycles of 96 °C for 10 s, 50 °C for 15 s, and 60 °C for 1 min.
Extension products were analyzed with the WAVE DNA Fragment Analysis System (Transgenomic), using a DNASep column kept at 70 °C in an oven. Extension products were denatured at 96 °C for 1 min and immediately placed on ice. Ten-microliter aliquots of the extension products were automatically injected into the DNASep column and eluted with a linear acetonitrile gradient in 0.1 mmol/L triethylammonium acetate buffer (pH 7.4) at a constant flow rate of 0.9 mL/min. The gradient was composed of 15–40% buffer B created over 10 min by mixing buffers A and B, where buffer A was 0.25 mL/L acetonitrile in 0.1 mol/L triethylammonium acetate and buffer B was 250 mL/L acetonitrile in 0.1 mol/L triethylammonium acetate. The column was cleaned after each analytical run with 750 mL/L acetonitrile, equilibrated, and returned to the gradient at 15% buffer B for 1 min. The eluate was monitored by an ultraviolet detector at 260 nm. Each batch of analytical runs was preceded by an injection of a mixture composed of the five primers only. This served to facilitate the identification of the primer peaks in the chromatograms by overlaying the primers-only chromatogram with that of a test sample.
Control DNA samples with known ß-globin genotypes (wild type or heterozygous for one of the five ß-thalassemia mutations, as determined by DNA sequencing) were studied to optimize the conditions for minisequencing and DHPLC and were needed for establishing the identity of the extension product peaks. For every mutation site analyzed, there were two (homozygous wild type or mutant) or three peaks (heterozygous; Fig. 1
). The primer peak always had the shortest retention time compared with its respective extension products. Our limited data (not shown) indicated that the extension products were retained in the order of C < G < T < A, which represent the 3' dideoxynucleotides of the products. These differ from the results (C < G < A < T) based on oligonucleotides with 3' deoxynucleotides (5)(6). The retention times were highly reproducible, with all CVs <0.70% except one (1.3%; Table 1
), and the control samples could be genotyped correctly and reproducibly. Thus, the presence of a particular mutation under study was indicated by the presence of a peak with a retention time characteristic of this mutation. This is the first study reporting the combined use of multiplex minisequencing and DHPLC for simultaneous genotyping of five ß-thalassemia mutations common in Southeast Asia.
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The advantages of minisequencing include the enhanced efficiency and specificity of Thermo Sequenase, accurate differentiation of heterozygosity and homozygosity within the same reaction, and the multiplexing capacity, as has been discussed by others (3)(7). Minisequencing reaction conditions are very robust, and the same reaction conditions can be used for any mutations without additional optimization. Multiplexing also allows genotyping of almost all known mutations of a given inherited disease in any given population with the use of only a few mutation panels, each consisting of five to eight mutations (7). Our experience indicates that multiplexing of seven to eight mutations on DHPLC can easily be achieved by adjusting the acetonitrile gradient and the rate of increase of buffer B. Compared with capillary electrophoresis (CE) for the detection of fluorescently labeled extension products (7), DHPLC offers the following advantages: use of unlabeled ddNTPs in minisequencing, direct analysis of extension products without purification, and short analytical run times (
11 min vs 25 min in CE). Size standards are not required in DHPLC, but are recommended in CE. The throughput can be further increased or even doubled if an integrated WAVE Accelerator (Transgenomic) is used. As in CE, multiple parallel analyses are also feasible with the multiple monolithic capillary columns (8), which, however, are not yet now commercially available.
Similarly, another level of multiplexing can also be added at extra cost by use of a fluorescence detector and ddNTPs labeled with different fluorophores. This allows easy identification of wild-type and mutant alleles because of the added dimension of fluorophore color. This does, of course, also mean further experimentation because different fluorophores affect retention differently (4). When these advantages are fully used, it is envisioned that multiplex minisequencing and DHPLC can be used to genotype ß-thalassemia mutations for a large number of samples at high speed and at a reasonable cost. In other words, this approach has the potential of being amenable to automation and being used in high-throughput screening. This potential is easily appreciated considering the widespread use of HPLC in routine clinical laboratories in the analysis of hemoglobin and other biomolecules of clinical significance. Most commonly used mutation detection methods, however, do not have this potential.
On the other hand, minisequencing-based methods always involve two rounds of amplification: one exponential (PCR) to generate the template for minisequencing and one linear (minisequencing reaction) to generate the extension products. This means longer overall analysis time. Because size and base composition influence the retention of single-stranded DNA in DHPLC (4), multiplexing of two or more minisequencing reactions requires empirical determination of the retention times for the primers and extension products in DHPLC. This means extensive and difficult experimentation during the development stage. As in CE, mobility does not always match the lengths of the extension products. Systematic studies should be conducted to investigate the effects of the 5' noncomplementary tails (in terms of length and base composition) of the minisequencing primers on the retention time. General principles developed from such studies can greatly facilitate future development of diagnostic mutation panels based on multiplex minisequencing and DHPLC for genotyping genetic diseases, particularly those showing extensive allelic heterogeneity, e.g., thalassemia (1) and cystic fibrosis (9). In other words, it is possible that such a protocol can be used for genotyping these diseases in routine diagnostic laboratories, particularly in areas where these diseases are prevalent.
Acknowledgments
This work was supported in part by the School of Nursing, The Hong Kong Polytechnic University. Purchase of the WAVE DNA Fragment Analysis System was supported by a Big Equipment Grant (G.53.27.9027) awarded to SPY by Hong Kong Polytechnic University. This work was presented at the Joint Annual Scientific Meeting 2003 organized by the Hong Kong Society of Haematology and the Hong Kong Association of Blood Transfusion and Haematology, March 29, 2003, Hong Kong SAR, China. We would also like to thank the reviewers for their constructive comments, which helped to improve the manuscript.
References
The following articles in journals at HighWire Press have cited this article:
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  K. Glynou, P. Kastanis, S. Boukouvala, V. Tsaoussis, P. C. Ioannou, T. K. Christopoulos, J. Traeger-Synodinos, and E. Kanavakis High-Throughput Microtiter Well-Based Chemiluminometric Genotyping of 15 HBB Gene Mutations in a Dry-Reagent Format Clin. Chem., March 1, 2007; 53(3): 384 - 391. [Abstract] [Full Text] [PDF]
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