title: DNA microarray-based solid-phase PCR on copoly (DMA-NAS-MAPS) silicon coated slides: An example of relevant clinical application.
abstract: In a previous study we developed a highly sensitive DNA microarray for the detection of common KRAS oncogenic mutations, which has been proven to be highly specific in assigning the correct genotype without any enrichment strategy even in the presence of minority mutated alleles. However, in this approach, the need of a spotter for the deposition of the purified PCR products on the substrates and the purification step of the conventional PCR are serious drawbacks. To overcome these limitations we have introduced the solid-phase polymerase chain reaction (SP-PCR) to form the array of PCR products starting from the oligonucleotide primers. This work was possible thanks to the great thermal stability of the copoly (DMA-NAS-MAPS) coating which withstands PCR thermal cycling temperatures. As an example of the application of this platform we performed the analysis of six common mutations in the codon 12 of KRAS gene (G12A, G12C, G12D, G12R, G12S, and G12V). In conclusion solid-phase PCR, combined with dual-color hybridization, allows mutation analysis in a shorter time span and is more suitable for automation.
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco, 9, 20131 Milano, Italy. Electronic address: francesco.damin@icrm.cnr.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Damin
authors.affiliation: Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Galbiati
authors.affiliation: Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy; Laboratory of Clinical Molecular Biology, IRCCS Ospedale San Raffaele, Italy; Università Vita-Salute San Raffaele, Milan, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Ferrari
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Chiari
mesh_terms: D015374:Biosensing Techniques; D005838:Genotype; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011108:Polymers; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000302:isolation & purification*; D012825:Silicon / Q000737:chemistry
chemical_list: C117307:KRAS protein, human; D011108:Polymers; D016283:Proto-Oncogene Proteins p21(ras); D012825:Silicon
keywords: DNA microarray; Genotyping; Polymer coated silicon slide; Solid-phase PCR