REAL-TIME PCR OF TARGETS ON A MICRO-ARRAY
First Claim
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1. A method for PCR amplification and detection of a polynucleotide molecule being present in a solution contained in a chamber having a surface bearing the capture molecules for the detection of the amplified sequences comprising:
- providing a rotating holder and a reaction chamber having fixed upon one of its surface at least a capture molecule being immobilized in a localized areas of a flat surface of said reaction chamber,introducing a solution containing said polynucleotide molecule into said reaction chamber and reagents for polynucleotide 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 polynucleotide molecule by PCR amplification, wherein the change in temperatures is obtained by changing the temperature of the air around the reaction chamber and wherein the reaction chamber is subjected to a rotating movement,performing at least a measurement of the labelled target polynucleotide molecule in the following way,incubating said labelled target polynucleotide molecule present in said solution under conditions allowing a specific hybridization between said target polynucleotide molecule and its corresponding capture molecule (20),measuring a signal originating from the surface having the bound labelled target polynucleotide molecule in response to illumination of said flat surface, wherein the signal is measured outside the chamber, and wherein the surface of signal emission comprises at least one localized area having a surface of between about 0.1 μ
m2 and about 75 mm2, wherein the said surface has a homogeneous interface during the measurement of the signal andprocessing the data obtained in order to detect and/or quantify the amount of polynucleotide molecule present in the solution before the amplification.
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Abstract
The present invention relates to a method, apparatus, cartridge and kit for monitoring on a micro-array a real-time PCR amplification of a polynucleotide molecule being present in a solution.
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Citations
99 Claims
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1. A method for PCR amplification and detection of a polynucleotide molecule being present in a solution contained in a chamber having a surface bearing the capture molecules for the detection of the amplified sequences comprising:
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providing a rotating holder and a reaction chamber having fixed upon one of its surface at least a capture molecule being immobilized in a localized areas of a flat surface of said reaction chamber, introducing a solution containing said polynucleotide molecule into said reaction chamber and reagents for polynucleotide 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 polynucleotide molecule by PCR amplification, wherein the change in temperatures is obtained by changing the temperature of the air around the reaction chamber and wherein the reaction chamber is subjected to a rotating movement, performing at least a measurement of the labelled target polynucleotide molecule in the following way, incubating said labelled target polynucleotide molecule present in said solution under conditions allowing a specific hybridization between said target polynucleotide molecule and its corresponding capture molecule (20), measuring a signal originating from the surface having the bound labelled target polynucleotide molecule in response to illumination of said flat surface, wherein the signal is measured outside the chamber, and wherein the surface of signal emission comprises at least one localized area having a surface of between about 0.1 μ
m2 and about 75 mm2, wherein the said surface has a homogeneous interface during the measurement of the signal andprocessing the data obtained in order to detect and/or quantify the amount of polynucleotide molecule present in the solution before the 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, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80)
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39. A method for PCR amplification and detection of a polynucleotide molecule being present in a solution contained in a chamber having a surface bearing the capture molecules for the detection of the amplified sequences comprising the steps of:
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providing a rotating holder (1) and a reaction chamber (2) having fixed upon one of its surface at least a capture molecule (20) being immobilized in a localized areas (21) of a flat surface of said reaction chamber, introducing a solution containing said polynucleotide molecule into said reaction chamber (2) and reagents for polynucleotide molecule amplification and labelling, submitting the solution to at least 2 thermal cycles comprising a denaturation, annealing and elongation steps in order to obtain labelled target polynucleotide molecule by PCR amplification, performing at least a measurement of the labelled target polynucleotide molecule in the following way, incubating said labelled target polynucleotide molecule present in said solution under conditions allowing a specific hybridization between said target polynucleotide molecule and its corresponding capture molecule (20), measuring a signal originating from the surface having the bound labelled target polynucleotide molecule in response to illumination of said flat surface, wherein the signal is measured outside the chamber, and wherein the surface of signal emission comprises at least one localized area having a surface of between about 0.1 μ
m2 and about 75 mm2, wherein the said surface has a homogeneous interface during the measurement of the signal,wherein the hybridization and the annealing steps occur at the same temperature and wherein the Tm of the primers for a target are within a range of the temperature of annealing plus 0-4°
C. and the Tm of the probe for said target is within a range of temperature of the hybridization plus 6-12°
C. andProcessing the data obtained in order to detect and/or quantify the amount of polynucleotide molecule present in the solution before the amplification.
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81. An apparatus for monitoring on a micro-array a PCR amplification of a polynucleotide molecule being present in a solution comprising:
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a rotating holder and a reaction chamber having fixed upon its surface a micro-array, comprising at least one capture molecule being immobilized in specifically localized areas of a flat surface of said chamber, which is in fluid communication in a chamber with said polynucleotide molecule and reagents for polynucleotide molecule amplification and labelling, a thermal cycler for carrying out an automated PCR process, said thermal cycler capable of changing the temperature of the air around the chamber for producing labelled target polynucleotide molecule, a rotor for rotating said holder and said reaction chamber, an illumination light source, a detector for measuring a signal from the bound labelled target polynucleotide molecule, with said solution being present in the chamber and containing the labelled target polynucleotide molecule wherein the surface of emission for a localized area is comprised between about 0.1 μ
m2 and about 75 mm2 and has a homogeneous interface,wherein the different parts are integrated into the same apparatus in order to read the signal of the bound labelled target polynucleotide molecule during the PCR amplification. - View Dependent Claims (82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95)
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96. A cartridge for monitoring on a micro-array a PCR amplification of a polynucleotide molecule being present in a solution, said cartridge comprising:
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a substrate (optic bloc) comprising a first flat surface and a flat second surface, said first flat surface comprising a micro-array comprising at least 20 different capture molecules being immobilized in specifically localized areas; and an airlock comprising an inlet port, a mounting surface and a reaction chamber (2);
said reaction chamber comprising a channel constructed to permit fluid flow from said inlet port into said reaction chamber, wherein said first flat surface of said substrate is mounted with respect to said mounting surface thereby covering said reaction chamber and whereby said micro-array is located inside said reaction chamber. - View Dependent Claims (97, 98)
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99. A kit for monitoring on a micro-array a PCR amplification of a polynucleotide molecule being present in a solution, said kit comprising a cartridge having a substrate (optic bloc) comprising a first flat surface and a flat second surface, said first flat surface comprising a micro-array comprising at least 20 different capture molecules being immobilized in specifically localized areas and primers for the amplification of target, wherein the Tm of the immobilized probes for said target is at least 4°
- C. and preferably 6°
C. higher than the Tm of the two primers specific of said target and a solution for the PCR including glutamate.
- C. and preferably 6°
Specification