Novel dna chips
First Claim
1. A method for detecting a mutation at position n in a target nucleic acid, characterized in that it comprises the following steps:
- a) hybridization of a probe linked in 5′
to a solid support of the DNA chip type with a target nucleic acid, the 3′
end of said probe hybridizing at most up to nucleotide n-1 of the target nucleic acid;
b) elongation of the probe hybridized in step a) by incorporation, in the 5′
-3′
direction of nucleotides complementary to said target nucleic acid by means of a reaction mixture comprising at least one nucleotide derivative resistant to degradation by an exonuclease and a DNA polymerase, c) digestion with said exonuclease such that only the probes elongated in step b) are not degraded, washing, d) detection of the presence or absence of mutation by directly or indirectly measuring the presence or absence of DNA.
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Abstract
The invention concerns a DNA chip system for detecting mutation in a target nucleic acid such that only the DNA comprising the mutation remains on the chip at the end of the process. The invention concerns a method which consists in adding a complementary αS-phosphothioatedesoxynucleotide of the mutation is added by means of DNA polymorase at the 3′ end of the probe hybridised with the target nucleic acid and in adding an exonuclease so that only the elongated probes are not degraded. The detection of the presence or absence of mutation is carried out by directly or indirectly measuring the presence or the absence of DNA in a specific site on the chip. Advantageously, the chip comprises ISFET transistors or piezoelectric transducers.
186 Citations
24 Claims
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1. A method for detecting a mutation at position n in a target nucleic acid, characterized in that it comprises the following steps:
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a) hybridization of a probe linked in 5′
to a solid support of the DNA chip type with a target nucleic acid, the 3′
end of said probe hybridizing at most up to nucleotide n-1 of the target nucleic acid;
b) elongation of the probe hybridized in step a) by incorporation, in the 5′
-3′
direction of nucleotides complementary to said target nucleic acid by means of a reaction mixture comprising at least one nucleotide derivative resistant to degradation by an exonuclease and a DNA polymerase,c) digestion with said exonuclease such that only the probes elongated in step b) are not degraded, washing, d) detection of the presence or absence of mutation by directly or indirectly measuring the presence or absence of DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A kit comprising a DNA chip to which there are attached probes and at least one of the elements chosen from:
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a batch of 4 reaction mixtures each comprising a different α
S-phosphothioatedexoynucleotide selected from α
S-dATP, α
S-dTTP, α
S-dCTP and α
S-dGTP, α
S-dUTP and α
S-dITP,a DNA polymerase, an exonuclease, in particular exonuclease III, a batch of solutions for solubilizing the DNA polymerase and/or the exonuclease in the case where these enzymes exist in the form of a powder. - View Dependent Claims (21, 22, 23, 24)
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Specification