Methods for amplifying and detecting multiple polynucleotides on a solid phase support
First Claim
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1. A method for detecting multiple target polynucleotides in a sample, the method comprising the steps of:
- a) providing an array of oligonucleotide primers wherein each array contains one or more groups of oligonucleotide primers immobilized on discrete areas of a solid phase support, each group of oligonucleotide primers selectable for a particular target polynucleotide and comprising primer sequences complementary to a sequence of the particular target polynucleotide;
b) contacting the array with a reaction mixture comprising a sample, said sample containing or suspected of containing multiple target polynucleotides, said reaction mixture further comprising reagents suitable for polynucleotide hybridization and amplification;
c) performing polymerase-mediated polynucleotide amplification for at least two rounds, whereby the target polynucleotides serve as initial templates for the synthesis of detectable nascent polynucleotides which are extended from the immobilized primers; and
d) detecting the presence and quantity of the synthesized polynucleotides which are captured on discrete areas of the solid phase support via corresponding immobilized primers.
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Abstract
Methods for solid phase polymerase-mediated amplification using immobilized primers on a microarray are provided for detecting and cloning multiple target polynucleotides. The methods, compositions and kits provided herein are useful for research and clinical applications, particularly for large scale assays of genetic information in biological samples of interest.
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Citations
33 Claims
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1. A method for detecting multiple target polynucleotides in a sample, the method comprising the steps of:
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a) providing an array of oligonucleotide primers wherein each array contains one or more groups of oligonucleotide primers immobilized on discrete areas of a solid phase support, each group of oligonucleotide primers selectable for a particular target polynucleotide and comprising primer sequences complementary to a sequence of the particular target polynucleotide;
b) contacting the array with a reaction mixture comprising a sample, said sample containing or suspected of containing multiple target polynucleotides, said reaction mixture further comprising reagents suitable for polynucleotide hybridization and amplification;
c) performing polymerase-mediated polynucleotide amplification for at least two rounds, whereby the target polynucleotides serve as initial templates for the synthesis of detectable nascent polynucleotides which are extended from the immobilized primers; and
d) detecting the presence and quantity of the synthesized polynucleotides which are captured on discrete areas of the solid phase support via corresponding immobilized primers. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method for detecting and comparing the expression patterns of multiple target polynucleotides from at least two different biological sources, the method comprising the steps of:
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a) providing an array of multiple groups of oligonucleotide primers immobilized on a solid phase support, each group of oligonucleotide primers being selected for a particular target polynucleotide and comprising primers complementary to a sequence of a target polynucleotide, wherein target polynucleotides from each biological source contain a covalently linked sequence tag unique to the biological source;
b) contacting the array with a sample comprising multiple target polynucleotides from at least two different biological sources;
c) performing a first round of polymerase-mediated polynucleotide amplification under conditions suitable for polynucleotide hybridization and amplification, whereby the target polynucleotides from the different biological sources serve as initial templates for synthesis of complementary nascent polynucleotide strands which are extended from the immobilized primers;
d) performing a second round of polymerase-mediated polynucleotide amplification in the presence of solution phase sequence tags that are unique to each biological source, wherein the solution phase sequence tags serve as primers and the immobilized nascent polynucleotide strands from step c) serve as templates for the synthesis of new amplification products which are extended from the solution phase sequence tags; and
e) detecting and comparing the immobilized amplification products of the target polynucleotides from the different biological sources. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. A kit for detecting multiple target polynucleotides in a sample, comprising:
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a) a solid phase support comprising a plurality of immobilized and separate populations of oligonucleotide primers each population selected to hybridize to a different target polynucleotide, wherein each primer is suitable for conducting a polymerase chain reaction with a specific target polynucleotide to amplify the polynucleotide;
b) reagents necessary for conducting a polymerase mediated amplification reaction on the solid support; and
c) detection means for detecting amplified polynucleotides on the support. - View Dependent Claims (28, 29)
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30. An array for detecting and comparing the expression patterns of multiple target polynucleotides from at least two different biological sources, the array comprising:
at least two groups of oligonucleotide primers immobilized on discrete areas of a solid phase support, each group of oligonucleotide primers being selected for a particular target polynucleotide and comprising primers complementary to a sequence of the particular target polynucleotide, wherein each group is identifiable by its position on the solid support and, further wherein each group comprises primers usable for detecting expression patterns of target polynucleotide from a single biological source. - View Dependent Claims (31, 32, 33)
Specification