Real-time PCR of targets on a micro-array
First Claim
1. A method for monitoring on a micro-array a PCR amplification of a nucleotide molecule being present in a solution comprising the steps of:
- providing a support having fixed upon its surface a micro-array comprising at least one capture molecule immobilized in specifically localized area(s) of said support and a reaction chamber,introducing a solution containing said nucleotide molecule into said reaction chamber and reagents for nucleotide molecule amplification and labeling,submitting the solution to at least five thermal cycles having at least 2 different temperature steps for denaturation, annealing and elongation in order to obtain labelled target nucleotide molecule by PCR amplification,measuring the labelled target nucleotide molecule during at least five thermal cycles of the PCR amplification in the following way;
incubating said labelled target nucleotide molecule under conditions allowing a specific binding between said target nucleotide molecule and its corresponding capture molecule,measuring light emission from the bound, labelled target nucleotide molecule in response to excitation light with said solution being present in the chamber and containing the labeled target nucleotide molecule, wherein the surface of emission for a localized area is between about 0.1 μ
m2 and about 75 mm2, and wherein said measurement is performed in real time during annealing and/or elongation steps of the at least five thermal cycles of the PCR amplification, andprocessing data obtained in the at least five thermal cycles of the PCR amplification in order to detect and/or quantify an amount of nucleotide molecule present in the solution during PCR amplification.
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Accused Products
Abstract
The present invention relates to a method and apparatus for monitoring on a micro-array a PCR amplification of a nucleotide molecule being present in a solution. The method includes the steps of: providing a support having fixed upon its surface a microarray having at least a capture molecule being immobilized in specifically localized areas of the support and a reaction chamber; introducing a solution containing the nucleotide molecule into the reaction chamber and reagents for nucleotide molecule amplification and labelling; submitting the solution to at least 2 thermal cycles having at least 2 and preferably 3 different temperature steps in order to obtain labelled target nucleotide molecule by PCR amplification; performing at least a measurement of the labelled target nucleotide molecule in at least one thermal cycle by incubating the labelled target nucleotide molecule under conditions allowing a specific binding between the target nucleotide molecule and its corresponding capture molecule and measuring the light emission from the bound labelled target nucleotide molecule in response to excitation light with the solution being present in the chamber and containing the labelled target nucleotide molecule. The surface of emission for a localized area is between about 0.1 μm2 and about 75 mm2. The method further includes processing the data obtained in at least one thermal cycle in order to detect and/or quantify the amount of nucleotide molecule present in the solution before the amplification.
6 Citations
31 Claims
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1. A method for monitoring on a micro-array a PCR amplification of a nucleotide molecule being present in a solution comprising the steps of:
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providing a support having fixed upon its surface a micro-array comprising at least one capture molecule immobilized in specifically localized area(s) of said support and a reaction chamber, introducing a solution containing said nucleotide molecule into said reaction chamber and reagents for nucleotide molecule amplification and labeling, submitting the solution to at least five thermal cycles having at least 2 different temperature steps for denaturation, annealing and elongation in order to obtain labelled target nucleotide molecule by PCR amplification, measuring the labelled target nucleotide molecule during at least five thermal cycles of the PCR amplification in the following way; incubating said labelled target nucleotide molecule under conditions allowing a specific binding between said target nucleotide molecule and its corresponding capture molecule, measuring light emission from the bound, labelled target nucleotide molecule in response to excitation light with said solution being present in the chamber and containing the labeled target nucleotide molecule, wherein the surface of emission for a localized area is between about 0.1 μ
m2 and about 75 mm2, and wherein said measurement is performed in real time during annealing and/or elongation steps of the at least five thermal cycles of the PCR amplification, andprocessing data obtained in the at least five thermal cycles of the PCR amplification in order to detect and/or quantify an amount of nucleotide molecule present in the solution during PCR amplification. - 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)
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Specification