Locus specific amplification using array probes
First Claim
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1. A method for amplifying and analyzing a plurality of target sequences from a nucleic acid sample, said method comprising:
- (a) fragmenting the nucleic acid sample with a restriction enzyme to obtain fragments with known sequences at the 5′ and
3′
ends of the fragments, wherein said fragments comprise a plurality of target fragments comprising target sequences;
(b) mixing the fragments obtained in (a) with a plurality of target specific splint oligonucleotides, wherein each splint oligonucleotide comprises a first target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at the 3′
end of a corresponding target fragment, and a second target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at and including the 5′
end of said corresponding target fragment, and wherein said first sequence is 5′
of said second sequence in said splint oligonucleotide, wherein target specific splint oligonucleotides further comprises a first common priming sequence at the 5′
end and a second common priming sequence at the 3′
end;
(c) adding first and second primers to the mixture wherein said first primer is complementary to said first common priming sequence and said second primer is complementary to said second common priming sequence and wherein said first and second primers hybridize to said splint oligonucleotides so the first primer is adjacent to the 3′
end of the target fragment and the second primer is adjacent to the 5′
end of the target fragment;
(d) ligating the first primer to the 3′
end of the target fragment and the second primer to the 5′
end of the target fragment to obtain ligated target fragments comprising a first common priming site at the 3′
end and a second common priming sites at the 5′
end;
(e) after said ligating step (d) fragmenting said splint oligonucleotides;
(f) amplifying the ligated target fragments from (d) to obtain amplified target fragments; and
(g) analyzing the amplified target fragments by a method comprising hybridization to an array comprising a plurality of oligonucleotide probes present at known or determinable locations in the array.
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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. In preferred aspects the analysis is a determination of sequence at one or more locations in the amplified target. The methods may be used for genotyping, sequencing and analysis of copy number.
167 Citations
21 Claims
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1. A method for amplifying and analyzing a plurality of target sequences from a nucleic acid sample, said method comprising:
-
(a) fragmenting the nucleic acid sample with a restriction enzyme to obtain fragments with known sequences at the 5′ and
3′
ends of the fragments, wherein said fragments comprise a plurality of target fragments comprising target sequences;(b) mixing the fragments obtained in (a) with a plurality of target specific splint oligonucleotides, wherein each splint oligonucleotide comprises a first target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at the 3′
end of a corresponding target fragment, and a second target complementary sequence that is at least 10 bases in length and is perfectly complementary to the at least 10 bases at and including the 5′
end of said corresponding target fragment, and wherein said first sequence is 5′
of said second sequence in said splint oligonucleotide, wherein target specific splint oligonucleotides further comprises a first common priming sequence at the 5′
end and a second common priming sequence at the 3′
end;(c) adding first and second primers to the mixture wherein said first primer is complementary to said first common priming sequence and said second primer is complementary to said second common priming sequence and wherein said first and second primers hybridize to said splint oligonucleotides so the first primer is adjacent to the 3′
end of the target fragment and the second primer is adjacent to the 5′
end of the target fragment;(d) ligating the first primer to the 3′
end of the target fragment and the second primer to the 5′
end of the target fragment to obtain ligated target fragments comprising a first common priming site at the 3′
end and a second common priming sites at the 5′
end;(e) after said ligating step (d) fragmenting said splint oligonucleotides; (f) amplifying the ligated target fragments from (d) to obtain amplified target fragments; and (g) analyzing the amplified target fragments by a method comprising hybridization to an array comprising a plurality of oligonucleotide probes present at known or determinable locations in the array. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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Specification
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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., Shapero, Michael H., Wang, Pei-Hua
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Primary Examiner(s)Boesen, Christian C
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Application NumberUS15/184,393Publication NumberTime in Patent Office1,104 DaysField of SearchNoneUS Class CurrentCPC Class CodesC12Q 1/6837 using probe arrays or probe...C12Q 1/6844 Nucleic acid amplification ...C12Q 1/6858 Allele-specific amplificationC12Q 2521/307 Single strand endonucleaseC12Q 2531/113 PCRC12Q 2537/143 Multiplexing, i.e. use of m...C12Q 2565/513 characterised by the patter...C40B 50/06 Biochemical methods, e.g. u...
