Amplification and analysis of selected targets on solid supports
First Claim
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1. A method for amplifying a plurality of target sequences from a nucleic acid sample and analyzing the amplified target sequences, the method comprising:
- (a) fragmenting the nucleic acid sample to obtain target fragments comprising target sequences;
(b) mixing the target fragments obtained in (a) with an array of probes arranged in features on a solid support, wherein each feature comprises multiple copies of a target specific array probe having a first probe region that is perfectly complementary to a first target region and a second probe region that is perfectly complementary to a second target region, wherein the first probe region is 5′
of the second probe region and wherein the first target region is 5′
of the second target region, and wherein the probes are attached to the solid support at the 5′
end of the probe, wherein target fragments hybridize to complementary target specific array probes to form probe;
target fragment complexes wherein the target fragment hybridizes to the target specific array probe so that the first target region is hybridized to the first probe region and the second target region is hybridized to the second probe region;
(c) joining a 5′
end of each target fragment to the 3′
end of the same target fragment by enzymatic ligation to form target circles wherein the 5′
end of each target fragment and the 3′
end of the same target fragment are joined by ligating the 5′
end of the target fragment to the 3′
end of an oligonucleotide and ligating the 3′
end of the target fragment to the 5′
end of the oligonucleotide and wherein the extended array probe comprises a plurality of copies of the complement of the oligonucleotide;
(d) extending the array probes using the target circles as a template in a rolling circle amplification reaction, thereby obtaining extended array probes comprising a plurality of copies of the complement of a plurality of the target fragment; and
(e) analyzing the copies.
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Abstract
Methods are provided for multiplexed amplification of selected targets and analysis of the amplified targets. In preferred aspects the amplification and analysis take place on the same solid support and preferably in a localized area such as a bead or a feature of an array. Targets are circularized by hybridization to probes followed by ligation of the ends of the target to form a closed circle. The targets are then used as template for extension of an array bound probe resulting in extended probes having multiple copies of the target. The extended probes can then be analyzed. The methods may be used for genotyping, sequencing and analysis of copy number.
75 Citations
19 Claims
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1. A method for amplifying a plurality of target sequences from a nucleic acid sample and analyzing the amplified target sequences, the method comprising:
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(a) fragmenting the nucleic acid sample to obtain target fragments comprising target sequences; (b) mixing the target fragments obtained in (a) with an array of probes arranged in features on a solid support, wherein each feature comprises multiple copies of a target specific array probe having a first probe region that is perfectly complementary to a first target region and a second probe region that is perfectly complementary to a second target region, wherein the first probe region is 5′
of the second probe region and wherein the first target region is 5′
of the second target region, and wherein the probes are attached to the solid support at the 5′
end of the probe, wherein target fragments hybridize to complementary target specific array probes to form probe;
target fragment complexes wherein the target fragment hybridizes to the target specific array probe so that the first target region is hybridized to the first probe region and the second target region is hybridized to the second probe region;(c) joining a 5′
end of each target fragment to the 3′
end of the same target fragment by enzymatic ligation to form target circles wherein the 5′
end of each target fragment and the 3′
end of the same target fragment are joined by ligating the 5′
end of the target fragment to the 3′
end of an oligonucleotide and ligating the 3′
end of the target fragment to the 5′
end of the oligonucleotide and wherein the extended array probe comprises a plurality of copies of the complement of the oligonucleotide;(d) extending the array probes using the target circles as a template in a rolling circle amplification reaction, thereby obtaining extended array probes comprising a plurality of copies of the complement of a plurality of the target fragment; and (e) analyzing the copies. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method for amplifying a plurality of target sequences from a nucleic acid sample and analyzing the amplified target sequences, the method comprising:
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(a) fragmenting the nucleic acid sample to obtain target fragments comprising target sequences; (b) mixing the target fragments obtained in (a) with an array of probes arranged in features on a solid support, wherein each feature comprises multiple copies of a target specific array probe having a first probe region that is perfectly complementary to a first target region and a second probe region that is perfectly complementary to a second target region, wherein the first probe region is 5′
of the second probe region and wherein the first target region is 5′
of the second target region, and wherein the probes are attached to the solid support at the 5′
end of the probe, wherein target fragments hybridize to complementary target specific array probes to form probe;
target fragment complexes wherein the target fragment hybridizes to the target specific array probe so that the first target region is hybridized to the first probe region and the second target region is hybridized to the second probe region;(c) extending the 3′
end of the target using a common section of the array probe as template and joining a 5′
end of each target fragment to the 3′
end of the same target fragment to form target circles;(d) extending the array probes using the target circles as a template in a rolling circle amplification reaction, thereby obtaining extended array probes comprising a plurality of copies of the complement of a plurality of the target fragment; and (e) analyzing the copies. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19)
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Current AssigneeAffymetrix, Inc. (Thermo Fisher Scientific Incorporated)
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Original AssigneeAffymetrix, Inc. (Thermo Fisher Scientific Incorporated)
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InventorsFu, Glenn K., McGall, Glenn H., Kuimelis, Robert G., Hu, Jing, Wang, Pei-Hua
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Primary Examiner(s)BOESEN, CHRISTIAN C
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Application NumberUS12/899,540Publication NumberTime in Patent Office1,308 DaysField of SearchNoneUS Class Current506/9CPC Class CodesC12Q 1/6837 using probe arrays or probe...C12Q 1/6844 Nucleic acid amplification ...C12Q 1/6858 Allele-specific amplificationC12Q 1/6874 involving nucleic acid arra...C12Q 1/6876 Nucleic acid products used ...C12Q 2521/307 Single strand endonucleaseC12Q 2531/113 PCRC12Q 2531/125 Rolling circleC12Q 2537/143 Multiplexing, i.e. use of m...C12Q 2565/513 characterised by the patter...C12Q 2600/106 Pharmacogenomics, i.e. gene...C12Q 2600/154 Methylation markersC12Q 2600/156 Polymorphic or mutational m...C40B 30/04 by measuring the ability to...C40B 50/06 Biochemical methods, e.g. u...
