Identification and/or quantification method of nucleotide sequence (s) elements specific of genetically modified plants on arrays
First Claim
1. A method for identifying a genetically modified plant by an identification and/or quantification of different and multiple nucleotide sequence elements corresponding to at least a portion of an exogenous nucleotide sequence integrated into the genome of the genetically modified plant, wherein said elements are present in a biological sample and wherein the method comprises the steps of:
- a) amplifying or copying a nucleotide sequence element or part of it into target nucleotide sequences, using primer pairs which are able to amplify the said nucleotide sequence element being homologous with nucleotide sequence elements present in at least two different genetically modified plants wherein said nucleotide sequence element is selected from the group consisting of the following promoters, terminators and/or markers sequence(s);
P35s, T-nos, nptII, pat, Cry1Ab and EPSPS, and wherein the length of the target nucleotide sequence is comprised between about 100 and about 200 bases;
b) putting into contact the obtained target nucleotide sequences with single stranded capture nucleotide sequences bound by a covalent link to the insoluble solid support at a specific location of the solid support surface;
c) detecting the binding of the target nucleotide sequences, by detecting a signal resulting from hybridization by complementary base pairing of the target nucleotide sequence and its corresponding capture nucleotide sequence at the specific location, wherein the capture nucleotide sequence(s) are bound to the insoluble solid support at a specific location according to an array, having a density of at least 4 different bound single stranded capture nucleotide sequence(s)/cm2 of solid support surface and wherein the capture nucleotide sequence comprises a sequence having between 10 and 200 bases, which allows a specific hybridization with the target nucleotide sequence to be detected and/or quantified;
d) repeating the steps a) to c) for a second, third, fourth or more different nucleotide sequence elements specific of the genetically modified plant, and e) constructing a genetic map based on the presence or absence of these different and multiple nucleotide sequence elements; and
f) identifying the genetically modified plant in the biological sample based upon the constructed genetic map.
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Abstract
The present invention is related to an identification and/or quantification method of several organisms among many other ones possibly present in the analyzed sample having homologous sequence(s) by the determination of the genetic map of the organism. The method combines a limited number of amplifications of target sequence(s) using common primer pairs and the recording upon an array for the presence of single signals resulting from the binding between the capture sequence(s) and their corresponding target sequence(s) and correlating the presence of said detected target sequence(s) to the identification of some genetic specific sequence(s) of said (micro)organism(s) referred as genetic elements and from there to the identification of the organism. The method and device according to the invention allow the easy identification/detection of a sequence specific of an organism among other homologous sequence(s) and possibly its quantification. The identification of the various targets from the initial organism if obtained after their binding on specific capture probes present on a support or substrate preferably in the form of an array. The identification of the amplified targets is obtained directly, after washing of possible contaminants (unbound sequence(s)), by detecting and possibly recording for one target, a single spot signal at one specific location, wherein said capture nucleotide sequence was previously bound and said identification of a target is not a result of a complex pattern of spots upon the microarray to be analyzed in order to identify one target as proposed in the system of the state of the art.
13 Citations
34 Claims
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1. A method for identifying a genetically modified plant by an identification and/or quantification of different and multiple nucleotide sequence elements corresponding to at least a portion of an exogenous nucleotide sequence integrated into the genome of the genetically modified plant, wherein said elements are present in a biological sample and wherein the method comprises the steps of:
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a) amplifying or copying a nucleotide sequence element or part of it into target nucleotide sequences, using primer pairs which are able to amplify the said nucleotide sequence element being homologous with nucleotide sequence elements present in at least two different genetically modified plants wherein said nucleotide sequence element is selected from the group consisting of the following promoters, terminators and/or markers sequence(s);
P35s, T-nos, nptII, pat, Cry1Ab and EPSPS, and wherein the length of the target nucleotide sequence is comprised between about 100 and about 200 bases;
b) putting into contact the obtained target nucleotide sequences with single stranded capture nucleotide sequences bound by a covalent link to the insoluble solid support at a specific location of the solid support surface;
c) detecting the binding of the target nucleotide sequences, by detecting a signal resulting from hybridization by complementary base pairing of the target nucleotide sequence and its corresponding capture nucleotide sequence at the specific location, wherein the capture nucleotide sequence(s) are bound to the insoluble solid support at a specific location according to an array, having a density of at least 4 different bound single stranded capture nucleotide sequence(s)/cm2 of solid support surface and wherein the capture nucleotide sequence comprises a sequence having between 10 and 200 bases, which allows a specific hybridization with the target nucleotide sequence to be detected and/or quantified;
d) repeating the steps a) to c) for a second, third, fourth or more different nucleotide sequence elements specific of the genetically modified plant, and e) constructing a genetic map based on the presence or absence of these different and multiple nucleotide sequence elements; and
f) identifying the genetically modified plant in the biological sample based upon the constructed genetic map. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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Specification