Abstract
To test the feasibility of developing a diagnostic microarray for a specific disease, we selected all pathogenic changes of the β-globin gene occurring at a frequency ⩾1% in the multi-ethnic Dutch population for analysis. A tagged single-base extension (SBE) approach was used to detect 19 different mutations causing β-thalassemia or abnormal hemoglobins. In the SBE reaction, the primers were elongated at the 3′site with a fluorescently labeled dideoxyribonucleotide triphosphate (ddNTP) complementary to the mutation, following tag hybridization to a glass or flow-through microarray. We compared the performance of a generic glass array and a porous system, by testing each mutation separately using heterozygous carriers and by screening a cohort of 40 unknown β-thalassemia carriers and patients. The results were verified by direct sequencing. The microarray system was able to detect 17 β-globin mutations simultaneously with >95% accuracy in a single SBE reaction. The flow-through array performed slightly better (96%), but the main advantages of the system included real-time data recording and a considerable time saving achieved through a reduced hybridization time.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Weatherall DJ, Clegg JB : The Thalassaemia Syndromes. Oxford: Blackwell Publishing; 1981, (3rd edn).
Thein SL : Beta-thalassaemia. Baillières Clin. Haematol. 1998; 11: 91–126.
Giordano PC, Harteveld CL, Heister AJGM et al: The molecular spectrum of beta-thalassemia and abnormal hemoglobins in the allochtonous and autochtonous Dutch population. Community Genet 1998; 1: 243–251.
Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvänen AC : Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res 1997; 7: 606–614.
Syvänen AC : From gels to chips: ‘Minisequencing’ primer extension for analysis of point mutations and single nucleotide polymorphisms. 1999 [Review]. Hum Mutat 1999; 13: 1–10.
Gerry NP, Witowski NE, Day J, Hammer RP, Barany G, Barany F : Universal DNA microarray method for multiplex detection of low abundance point mutations. J Mol Biol 1999; 292: 251–262.
Fortina P, Delgrosse K, Sakamuze T et al: Simple two-color array-based approach for mutation detection. Eur J Hum Genet 2000; 8: 884–894.
Gemignani F, Perra C, Landi S et al: Reliable Detection of β-Thalassemia and G6PD Mutations by a DNA Microarray. Clin Chem 2002; 48: 2051–2054.
Hacia JG, Brody LC, Chee MS, Fodor SP, Collins FS : Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluorescence analysis. Nat Genet 1996; 14: 441–447.
Wang DG, Fan JB, Siao CJ et al: Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 1998; 15 280: 1077–1082.
Syvänen AC, Aalto-Setälä K, Harju L, Kontula K, Söderlund H : A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 1990; 8: 684–692.
Sokolov BP : Primer extension technique for the detection of single nucleotides in genomic DNA. Nucleic Acids Res 1990; 18: 3671.
Kuppuswami MN, Hoffmann JW, Kasper CK, Spitzer SG, Groce SL : Single-nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes. Proc Natl Acad Sci USA 1991; 88: 1143–1147.
Nikiforov TT, Rendle RB, Goelet P et al.: Genetic bit analysis: a solid-phase method for typing single nucleotide polymorphisms. Nucleic Acids Res 1994; 22: 4167–4175.
Fan J-B, Chen X, Halushka MK et al: Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays. Genome Res 2000; 10: 853–860.
Pastinen T, Raitio M, Lindroos K, Tainola P, Peltonen L, Syvänen AC : A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays. Genome Res 2000; 10: 1031–1042.
Beuningen R van, van Damme H, Boender P, Bastiaensen N, Chan A, Kievits T : Fast and specific hybridization using flow-through microarrays on porous metal oxide. Clin Chem 2001; 47: 1931–1933.
Miller SA, Dykes DD, Polesky HF : A simple salting-out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215.
Losekoot M, Fodde R, Harteveld CL, van Heeren H, Giordano PC, Bernini LF : Denaturing gradient gel electrophoresis and direct sequencing of PCR amplified genomic DNA: a rapid and reliable diagnostic approach to beta-thalassaemia. Br J Haematol 1990; 76: 269–274.
Beier M, Hoheisel JD : Versatile derivatisation of solid support media for covalent bonding on DNA-microchips. Nucleic Acids Res 1999; 27: 1970–1977.
SantaLucia Jr J : A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA. 1998; 95: 1460–1465.
Cai SP, Wall J, Kan YW, Chehab FF : Reverse dot blot probes fot the screening of β-thalassemia mutations in Asians and American Blacks. Hum Mutat 1994; 3: 59–63.
Old JM, Varawella NY, Weatherall DJ : Rapid detection and prenatal diagnosis of β-thalassemia: studies in Indian and Cypriot populations in the UK. Lancet 1990; 336: 834–837.
Acknowledgements
The authors thank Victor de Jager for optimization of the glass-coating protocol and Majid Yavarian for providing the Iranian samples.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
van Moorsel, C., van Wijngaarden, E., Fokkema, I. et al. β-Globin mutation detection by tagged single-base extension and hybridization to universal glass and flow-through microarrays. Eur J Hum Genet 12, 567–573 (2004). https://doi.org/10.1038/sj.ejhg.5201192
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/sj.ejhg.5201192
Keywords
This article is cited by
-
Development of a fibrous DNA chip for cost-effective β-thalassemia genotyping
International Journal of Hematology (2012)
-
Development of a Flow-Trough Microarray based Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Assay for the Detection of European Bunyaviruses
Molecular Biotechnology (2011)
-
TAMGeS: a Three-Array Method for Genotyping of SNPs by a dual-colour approach
BMC Genomics (2007)
-
Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA
BMC Genomics (2006)
