Patent FR-2920159-A1 (FR)
Title: Identifying, analyzing and/or quantifying alleles of polymorphisms in nucleic acid sample, comprises amplifying double strand of nucleic acid sequence, eliminating heteroduplexes, hybridizing products, and determining and comparing signal
Inventor: VALAT CHRISTOPHE
IPC: C12Q1/68
Filed: 2007-08-20
Abstract: Identifying, analyzing and/or quantifying alleles of polymorphisms in a nucleic acid sample of a subject, comprises: amplifying a double strand of a nucleic acid sequence comprising the polymorphism, and marking of amplicons; selectively eliminating heteroduplexes and digesting the second strand of homoduplexes; contacting and hybridizing the products with oligonucleotide probes immobilized on a solid support; determining the signal emitted by the marked strands hybridized on the probes; and comparing the signal determined in determining step and signal of a reference sample. Identifying, analyzing and/or quantifying alleles of one or more polymorphisms in a nucleic acid sample of a subject, comprises: amplifying a double strand of a nucleic acid sequence comprising the polymorphism for each polymorphism to be analyzed by the sample, and marking of amplicons produced at an end of one of the two produced strands either a double stranded amplicon of which a first strand is marked at its 5'end and a second strand is phosphorylated in 5', or a double stranded amplicon of which a first strand is marked at its 3'end and has 5'-hydroxy end, and the second strand is phosphorylated in 5'; denaturizing and hybridizing of amplicons obtained in the amplifying step; selectively eliminating heteroduplexes comprising a mismatched region formed in denaturizing step and digesting the second strand of homoduplexes by a specific exonuclease of a 5'-phosphate end; contacting and hybridizing the products of eliminating step with oligonucleotide probes immobilized on a solid support, where the probes comprise a probe for each analyzed polymorphism complementary of a part of first strand marked by amplicon corresponding to particular analyzed polymorphism; determining the signal emitted by the marked strands hybridized on the oligonucleotide probes; and comparing the signal determined in determining step and the signal of a reference sample, thus allowing the identification, analysis and/or quantification of alleles of one or more polymorphisms. Independent claims are included for: (1) a method for determining the identity of the nucleic acid sample in a reference sample, comprising determining homozygous or heterozygous character for one or more polymorphisms in the sample and to identify the reference sample, and comparing the homozygous or heterozygous character for polymorphisms tested in two samples, and the identity of the homozygous or heterozygous character to deduce the identity between the sample to identify and reference sample; and (2) a biochip comprising the oligonucleotide probes immobilized on the solid support, where the probes comprise a probe for each analyzed polymorphism complementary to a part of the first marked strand of amplicon corresponding to particular polymorphism analyzed by the sample of the nucleic acid of a subject to be treated.