High throughput genome sequencing on DNA arrays
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/68
C12P-019/34
C12M-001/36
출원번호
US-0981761
(2007-10-31)
등록번호
US-8440397
(2013-05-14)
발명자
/ 주소
Drmanac, Radoje
Callow, Matthew J.
Drmanac, Snezana
출원인 / 주소
Callida Genomics, Inc.
대리인 / 주소
Kilpatrick Townsend & Stockton LLP
인용정보
피인용 횟수 :
8인용 특허 :
140
초록▼
The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The inventi
The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both “upstream” and “downstream” of the adaptors, identification of entire target sequences may be accomplished.
대표청구항▼
1. A method of identifying a nucleotide at a detection position of a target nucleic acid, wherein said target nucleic acid comprises a plurality of detection positions, said method comprising: (a) providing a plurality of concatemers immobilized on a surface, wherein said surface comprises a plurali
1. A method of identifying a nucleotide at a detection position of a target nucleic acid, wherein said target nucleic acid comprises a plurality of detection positions, said method comprising: (a) providing a plurality of concatemers immobilized on a surface, wherein said surface comprises a plurality of discrete regions and said plurality of concatemers are randomly disposed on said discrete regions such that at least 70 percent of said concatemers are individually resolvable and such that at least 70 percent of said concatemers have a nearest neighbor distance of 200 nm or greater, wherein each concatemer comprises multiple copies of a monomer and each monomer comprises:(i) a first target domain of said target nucleic acid comprising a first set of target detection positions;(ii) a first adaptor;(iii) a second target domain of said target nucleic acid comprising a second set of target detection positions; and(iv) a second interspersed adaptor, wherein said second interspersed adaptor is located between said first target domain and said second target domain, and wherein said first target domain and said second target domain are contiguous segments of said target nucleic acid; (b) detecting a nucleotide in said first set or said second set of detection positions, thereby identifying said nucleotide. 2. The method of claim 1, wherein said target nucleic acid is genomic DNA. 3. The method of claim 1, wherein said monomer is formed by a method comprising: (a) providing a first circular polynucleotide comprising said first adaptor and a fragment of said target nucleic acid, wherein said first adaptor comprises a recognition site for a restriction enzyme;(b) cleaving said first circular polynucleotide with said restriction enzyme to form a first linear construct, wherein said restriction enzyme binds to said recognition site within said first adaptor and cleaves said fragment at a distance from said recognition site, wherein said first linear construct comprises said first adaptor interposed between said first target domain and said second target domain of said target nucleic acid;(c) ligating said second interspersed adaptor to said first linear construct to form a second linear construct;(d) circularizing said second linear construct to create a second circular polynucleotide, wherein said second circular polynucleotide comprises said first adaptor, said first target domain, said second interspersed adaptor, and said second target domain in that order. 4. The method of claim 3, wherein said restriction enzyme cleaves said fragment at a distance of 2 to 20 nucleotides from said recognition site. 5. The method of claim 3, wherein said restriction enzyme cleaves said fragment at a distance of 18 to 20 nucleotides from said recognition site. 6. The method of claim 1, wherein said first adaptor and said second interspersed adaptor have a known sequence and wherein said first and second target domains are unknown genomic sequences. 7. A method of identifying a nucleotide sequence of a target nucleic acid, said method comprising: (a) providing a plurality of concatemers randomly disposed on a surface, wherein said surface comprises a plurality of discrete regions and said plurality of concatemers are randomly disposed on said discrete regions such that at least 70 percent of said concatemers are individually resolvable and such that at least 70 percent of said concatemers have a nearest neighbor distance of 200 nm or greater, and wherein said concatemers comprise a multiple copies of a monomer and each monomer comprises: (i) a first sequence of said target nucleic acid,(ii) a second sequence of said target nucleic acid;(iii) a first interspersed adaptor located between said first sequence and said second sequence, wherein said first interspersed adaptor comprises at least one anchor probe hybridization site, and wherein said first interspersed adaptor is inserted into said target nucleic acid such that said first sequence and said second sequence are contiguous segments of said target nucleic acid;(b) hybridizing one or more anchor probes to said concatemers to form duplexes between said anchor probes and said anchor probe hybridization sites;(c) hybridizing one or more sequencing probes to said concatemers to form perfectly matched duplexes between said one or more sequencing probes and said first sequence or said second sequence of said target nucleic acid;(d) ligating anchor probes and sequencing probes that are hybridized to adjacent positions of said concatemers to form at least one ligation product; and(e) detecting said at least one ligation product, thereby identifying at least one nucleotide of said first sequence or said second sequence. 8. The method of claim 7, wherein said at least one anchor probe hybridization site is adjacent or close to the 3′ end of the second sequence. 9. The method of claim 7, wherein said at least one anchor probe hybridization site is adjacent or close to the 5′ end of the first sequence. 10. The method of claim 7, wherein said interspersed adaptor comprises two anchor probe hybridization sites, one adjacent or close to the 5′ end of said first sequence of the target nucleic acid and one adjacent or close to the 3′ end of said second sequence. 11. The method of claim 7, wherein said concatemers are attached to said discrete regions through a noncovalent interaction. 12. The method of claim 7, wherein each of said discrete regions has an area of less than 1 μm2. 13. The method of claim 7, wherein prior to said detecting step (e), said method further comprises removing any non-ligated anchor probes and sequencing probes. 14. The method of claim 7, wherein steps (b) through (e) are repeated a number of times. 15. The method of claim 14, wherein for each repetition of steps (b) through (e), said anchor probe hybridizes to a position shifted by at least one nucleotide from its position in the previous hybridization cycle, such that nucleotides in said first or second fragment are detected two or more times. 16. The method of claim 7, wherein said sequencing probes comprise a detectable label, and wherein said detecting step (e) comprises detecting said label. 17. The method of claim 7, wherein said first interspersed adaptor has a known sequence and wherein said first and second sequences are unknown genomic sequences. 18. The method of claim 1 or 7, wherein said surface further comprises inert inter-regional areas between said discrete regions and wherein said concatemers do not bind to said inert inter-regional areas. 19. The method of claim 1 or 7, wherein said discrete regions have a size from about 125 nm to about 250 nm. 20. The method of claim 1 or 7, wherein said discrete regions have a size from about 200 nm to about 500 nm. 21. The method of claim 1 or 7, wherein substantially all of said discrete regions have at most a single concatemer attached thereto. 22. A method of identifying a nucleotide at a detection position of a target nucleic acid, said method comprising: (a) providing a substrate comprising a surface, said surface comprising a plurality of immobilized concatemers, wherein each concatemer is formed by replication of a monomer such that said concatemer comprises multiple copies of said monomer, and wherein: (i) said monomer is formed from a target nucleic acid in which a first interspersed adaptor is inserted in the interior of said target nucleic acid, such that said first interspersed adaptor interrupts said target nucleic acid to form a first target sequence and a second target sequence;(ii) said first and second target sequence are contiguous segments of said target nucleic acid; and(b) detecting a nucleotide in said first set or said second target sequence, thereby identifying said nucleotide. 23. The method of claim 1, 7 or 22, wherein at least 70 percent of said concatemers have a nearest neighbor distance of 500 nm or greater. 24. The method of claim 1, 7 or 22, wherein at least 70 percent of said concatemers have a nearest neighbor distance of 700 nm or greater. 25. The method of claim 22, wherein said first interspersed adaptor has a known sequence and wherein said first and second target sequences are unknown genomic sequences.
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