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1. (WO2018178943) GENOTYPING OF MUTATIONS BY COMBINATION OF IN-TUBE HYBRIDIZATION AND UNIVERSAL TAG-MICROARRAY

Pub. No.:    WO/2018/178943    International Application No.:    PCT/IB2018/052219
Publication Date: Fri Oct 05 01:59:59 CEST 2018 International Filing Date: Sat Mar 31 01:59:59 CEST 2018
IPC: C12Q 1/6827
C12Q 1/6837
Applicants: CHIARI, Marcella
DAMIN, Francesco
GALBIATI, Silvia
FERRARI, Maurizio
Inventors: CHIARI, Marcella
DAMIN, Francesco
GALBIATI, Silvia
FERRARI, Maurizio
Title: GENOTYPING OF MUTATIONS BY COMBINATION OF IN-TUBE HYBRIDIZATION AND UNIVERSAL TAG-MICROARRAY
Abstract:
An assay method for a gene fragment of interest comprising: amplify at least one gene fragment of interest comprising or potentially comprising at least one SNP region of interest to form an initial amplification product; forming at least one single strand amplification product from the initial amplification product, wherein the single strand amplification product either comprises or potentially comprises the at least one SNP region of interest; hybridize in solution the single strand amplification product with at least one reporter molecule which comprises at least two, different domains of oligonucleotide, wherein a first domain of oligonucleotide is for hybridization with the single strand amplification product, and wherein the second domain of oligonucleotide is for hybridization to at least one microarray probe surface, wherein the microarray probe surface comprises at least one capture probe to allow for hybridization; contact the solution of the hybridized single strand amplification product with the at least one microarray probe surface which comprises at least one capture probe to allow for hybridization; detect for the presence of the hybridized single strand amplification product on the microarray surface. In the preferred embodiment relevant to cancer genotyping, the unexpectedly good results for KRAS oncogene indicate that all the seven codon 12 and 13 mutations studied could be unambiguously detected in less than 90 minutes in tissue clinical samples. Moreover, this system could reveal mutant alleles representing less than 0.1% of the starting material. By decoupling the mutation detection from the array hybridization, this technology becomes universal.