TY - JOUR
T1 - β-Thalassemia Microelectronic Chip
T2 - A Fast and Accurate Method for Mutation Detection
AU - Foglieni, Barbara
AU - Cremonesi, Laura
AU - Travi, Maurizio
AU - Ravani, Anna
AU - Giambona, Antonino
AU - Rosatelli, Maria Cristina
AU - Perra, Chiara
AU - Fortina, Paolo
AU - Ferrari, Maurizio
PY - 2004/1
Y1 - 2004/1
N2 - Background: β-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the β-thalassemia alleles in the Mediterranean area. Methods: We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, -87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes. Results: Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip. Conclusions: The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing β-thalassemia in other regions of the world.
AB - Background: β-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the β-thalassemia alleles in the Mediterranean area. Methods: We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, -87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes. Results: Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip. Conclusions: The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing β-thalassemia in other regions of the world.
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U2 - 10.1373/clinchem.2003.023077
DO - 10.1373/clinchem.2003.023077
M3 - Article
C2 - 14709638
AN - SCOPUS:1642492777
VL - 50
SP - 73
EP - 79
JO - Clinical Chemistry
JF - Clinical Chemistry
SN - 0009-9147
IS - 1
ER -