title: beta-Globin mutation detection by tagged single-base extension and hybridization to universal glass and flow-through microarrays.
abstract: To test the feasibility of developing a diagnostic microarray for a specific disease, we selected all pathogenic changes of the beta-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 beta-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 beta-thalassemia carriers and patients. The results were verified by direct sequencing. The microarray system was able to detect 17 beta-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.
authors.affiliation: Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Coline H M
authors.initials: CH
authors.lastname: van Moorsel
authors.forename: Erwin E
authors.initials: EE
authors.lastname: van Wijngaarden
authors.forename: Ivo F A C
authors.initials: IF
authors.lastname: Fokkema
authors.forename: Johan T
authors.initials: JT
authors.lastname: den Dunnen
authors.forename: Dirk
authors.initials: D
authors.lastname: Roos
authors.forename: Rob
authors.initials: R
authors.lastname: van Zwieten
authors.forename: Piero C
authors.initials: PC
authors.lastname: Giordano
authors.forename: Cornelis L
authors.initials: CL
authors.lastname: Harteveld
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D005006:Ethnicity / Q000235:genetics; D005091:Exons / Q000235:genetics; D005260:Female; D005914:Globins / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009426:Netherlands; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016324:Sequence Tagged Sites; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000208:ethnology / Q000235:genetics
chemical_list: D005914:Globins