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Development of new substrates for high‐sensitive genotyping of minority mutated alleles
Author(s) -
Galbiati Silvia,
Damin Francesco,
Di Carlo Gabriele,
Ferrari Maurizio,
Cremonesi Laura,
Chiari Marcella
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800242
Subject(s) - genotyping , allele , genetics , biology , genotype , gene
An unsurpassed level of sensitivity was reached in the detection of minority mutated alleles. A low‐density microarray was printed on a substrate specifically designed to provide an interference effect which amplifies the collection of the light emitted on the support and reinforces the intensity of excitation light. Optimal performance of the array was obtained by maximizing the probe density and the binding efficiency to the target through a polymeric coating made by the adsorption of a copolymer of N , N ‐dimethylacrylamide (97% of moles), N , N ‐acryloyloxysuccinimide (2%) and 3‐(trimethoxysilyl)propyl methacrylate (1%) synthesized by free radical copolymerization. The new substrate was used in the identification of fetal mutations in the maternal plasma DNA. Amino‐modified amplicons from genomic DNA corresponding to the locus of eight β‐thalassemia mutations were immobilized and interrogated with dual‐color oligonucleotide targets. Compared with the conventional glass substrates, the new substrate showed a great enhancement of fluorescence signals thanks to the combination of the optics with the highly efficient polymeric coating, allowing specific detection of all mutations. The high sensitivity and selectivity obtained made it possible to develop assays for the identification of paternally inherited mutations on fetal DNA in the maternal plasma in couples at risk for β‐thalassemia.