[특허]Methods for genotyping

Methods for genotyping 원문보기

IPC분류정보
국가/구분	United States(US) Patent 등록
국제특허분류(IPC7판)	C12Q-001/68
출원번호	US-0614948 (2006-12-21)
등록번호	US-9388459 (2016-07-12)
발명자 / 주소	Shapero, Michael H.
출원인 / 주소	Affymetrix, Inc.
대리인 / 주소	Banner & Witcoff, Ltd.
인용정보	피인용 횟수 : 0 인용 특허 : 103

초록 ▼

The present invention provides for methods for discriminating between alleles at polymorphic positions in a genome. In general the methods employ allele specific extension of oligonucleotides that are complementary to one of the alleles at the 3′ end of the oligonucleotide. The allele specific oligonucleotides are resistant to proof reading activity from a polymerase and may be extended in an allele specific manner by a DNA polymerase with a functional 3′ to 5′ exonuclease activity. The allele specific oligonucleotides may be attached to a solid support such as a chip or a bead.

대표청구항 ▼

1. A method for genotyping a plurality of polymorphisms in a nucleic acid sample, where each polymorphism has a first and a second allele, comprising: amplifying the nucleic acid sample using random primers and DNA polymerase in an isothermal amplification reaction to obtain an amplified nucleic acid sample;fragmenting the amplified nucleic acid sample to obtain a fragmented amplified nucleic acid sample;incubating the fragmented, amplified nucleic acid sample with a plurality of beads attached to allele-specific capture probes to allow formation of complexes between target fragments in the fragmented, amplified nucleic acid sample and allele-specific capture probes, wherein the plurality of beads are attached to a collection of allele-specific capture probes comprising at least 5,000different allele-specific capture probes, wherein the allele-specific capture probes comprise: (i) a linker,(ii) a cleavage region,(iii) a tag region, wherein each different allele-specific capture probe has a different sequence tag region and wherein the tag region is at least 15 bases; and,(iv) a target-specific region that terminates at its 3′ end with a base that is complementary to a polymorphic base in the target, wherein each different allele-specific capture probe has a different target-specific region;extending the allele-specific capture probes in the presence of labeled nucleotides using the target fragment as template to obtain labeled allele-specific capture probes, wherein extension of the allele-specific capture probes is blocked if there is a mismatch between the polymorphic position and the 3′ end of the allele-specific capture probe;separating the target fragment from the extended labeled allele-specific capture probes;cleaving the extended labeled allele-specific capture probes from the beads to produce released extended labeled allele-specific capture probes;detecting the released extended labeled allele-specific capture probes by hybridization to an array of tag probes of known sequence, wherein no amplification of the released extended labeled allele-specific capture probes occurs between the cleaving and detecting steps, wherein said tag probes are present at known or determinable locations on said array and each tag probe is complementary to a different tag region present in the allele-specific capture probes; anddetermining the genotype of said plurality of polymorphisms by determining which alleles are present, wherein the presence of hybridized labeled allele-specific capture probes is indicative of the presence of a particular allele in the nucleic acid sample. 2. The method of claim 1 wherein said step of cleaving comprises an enzymatic step. 3. The method of claim 2 wherein the cleavage region comprises one or more uracil bases and cleavage comprises incubation with uracil DNA glycosylase to generate abasic sites and the abasic sites are cleaved with an AP endonuclease or with acid or heat treatment. 4. The method of claim 2 wherein the cleavage region comprises one or more inosines and cleavage comprises incubation with Endonuclease V. 5. The method of claim 1 wherein said step of cleaving comprises photocleavage of a light-sensitive linkage in a capture probe. 6. A method for genotyping a plurality of polymorphisms in a nucleic acid sample comprising: amplifying the nucleic acid sample using random primers and DNA polymerase in an isothermal amplification reaction to obtain an amplified nucleic acid sample;fragmenting the amplified nucleic acid sample to generate target fragments;hybridizing a collection of capture probes comprising at least 5,000 different capture probes, to target fragments, wherein said capture probes are attached to a solid support at a 5′ end and each comprises:(i) a spacer sequence near said 5′ end,(ii) a dU region comprising a plurality of uracil residues,(iii) a tag sequence of at least 15 bases that is unique for each species of capture probe,(iv) a target-specific sequence, and(v) an allele-specific nucleotide corresponding to one allele of a polymorphism in said plurality of polymorphisms, wherein the capture probes terminate at their 3′ end with said allele-specific nucleotide;extending said capture probes with a DNA polymerase to generate extended capture probes in an allele-specific extension reaction;washing the solid support to remove the target fragments;cleaving the extended capture probes from the solid support by a method comprising photocleavage or enzymatic cleavage to produce released extended capture probes;detecting the released extended capture probes by hybridization to an array comprising a plurality of tag probe features, wherein no amplification of the released extended allele-specific capture probes occurs between the cleaving and detecting steps, wherein each tag probe feature comprises a different tag probe and wherein said tag probes are complementary to the tag sequences of the capture probes; anddetermining the genotype of said plurality of polymorphisms by determining which alleles are present, wherein the presence of hybridized allele-specific capture probes is indicative of the presence of a particular allele in the nucleic acid sample. 7. The method of claim 6 wherein the solid support comprises a plurality of beads. 8. The method of claim 7 wherein the beads are coated with anti-digoxidenin, and the capture probes comprise a digoxigenin label. 9. The method of claim 6 wherein the capture probes are exonuclease-resistant at the 3′ end and the DNA polymerase used to generate the extended capture probes has 3′ to 5′ exonuclease proofreading activity. 10. The method of claim 9 wherein the DNA polymerase is selected from the group consisting of T4 DNA polymerase, E. coli Klenow fragment, and T7 DNA polymerase. 11. The method of claim 6 wherein the cleavage from the solid support is enzymatic and comprises cleavage with an endonuclease. 12. The method of claim 6 wherein the cleavage from the solid support is enzymatic and comprises treatment with uracil DNA glycosylase and heat or acid treatment. 13. The method of claim 6, wherein the cleavage from the solid support is photocleavage and comprises exposure to UV light with a wavelength between 200 and 400 nanometers. 14. The method of claim 6 wherein the dU region comprises a plurality of inosine residues and the cleavage step comprises cleavage with Endonuclease V. 15. The method of claim 14 wherein the dU region comprises UIUI. 16. The method of claim 6 wherein the 3′ end of the capture probes comprises 0, 1, or 3 phosphorothioate linkages.

이 특허에 인용된 특허 (103)

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IPC	Description
A	생활필수품
A62	인명구조; 소방(사다리 E06C)
A62B	인명구조용의 기구, 장치 또는 방법(특히 의료용에 사용되는 밸브 A61M 39/00; 특히 물에서 쓰이는 인명구조 장치 또는 방법 B63C 9/00; 잠수장비 B63C 11/00; 특히 항공기에 쓰는 것, 예. 낙하산, 투출좌석 B64D; 특히 광산에서 쓰이는 구조장치 E21F 11/00)
A62B-1/08	.. 윈치 또는 풀리에 제동기구가 있는 것

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 국가코드, 자료구분, 상태, 출원번호, 출원일자, 공개번호, 공개일자, 등록번호, 등록일자, 발명명칭(한글), 발명명칭(영문), 출원인(한글), 출원인(영문), 출원인코드, 대표IPC 관리번호, 국가코드, 자료구분, 상태, 출원번호, 출원일자, 공개번호, 공개일자, 공고번호, 공고일자, 등록번호, 등록일자, 발명명칭(한글), 발명명칭(영문), 출원인(한글), 출원인(영문), 출원인코드, 대표출원인, 출원인국적, 출원인주소, 발명자, 발명자E, 발명자코드, 발명자주소, 발명자 우편번호, 발명자국적, 대표IPC, IPC코드, 요약, 미국특허분류, 대리인주소, 대리인코드, 대리인(한글), 대리인(영문), 국제공개일자, 국제공개번호, 국제출원일자, 국제출원번호, 우선권, 우선권주장일, 우선권국가, 우선권출원번호, 원출원일자, 원출원번호, 지정국, Citing Patents, Cited Patents
저장형식	Text(ASCII format) Excel format PIAS분석(.xls)
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

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Methods for genotyping 원문보기

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연합인증

Methods for genotyping 원문보기

초록 ▼

대표청구항 ▼

연구과제 타임라인

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이 특허에 인용된 특허 (103)

관련 콘텐츠

특허 원문 보기

IPC 상위 출원인

AI-Helper ※ AI-Helper는 오픈소스 모델을 사용합니다.

선택된 텍스트