US5695934A
Expired / Lapsed
US
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Massively parallel sequencing of sorted polynucleotides
Assignee: Lynx Therapeutics Inc
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Filing: 1994-12-19
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Granted: 1997-12-09
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Expires: 2014-12-19
Abstract
Abstract The invention provides a method and materials for sorting polynucleotides with oligonucleotide tags. Oligonucleotide tags of the invention are capable of hybridizing to complementary oligomeric compounds consisting of subunits having enhanced binding strength and specificity as compared to natural oligonucleotides. Such complementary oligomeric compounds are referred to herein as "tag complements." Subunits of tag complements may consist of monomers of non-natural nucleotide analogs, referred to herein as "antisense monomers" or they may comprise oligomers having lengths in the range of 3 to 6 nucleotides or analogs thereof, including antisense monomers, the oligomers being selected from a minimally cross-hybridizing set. In such a set, a duplex made up of an oligomer of the set and the complement of any other oligomer of the set contains at least two mismatches. Preferred antisense monomers include peptide nucleic acid monomers and nucleoside phosphoramidates having a 3'-NHP(O)(O--)O-5' linkage with its adjacent nucleoside. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports. This embodiment provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, mRNA fingerprinting, or the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.
Inventors
- 👤 Sydney Brenner
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 16:17
CPC Classifications
B; B01; B01J; B01J19/00; B01J19/0046; C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1065; C12N15/11; C12Q; C12Q1/00; C12Q1/68; C12Q1/6809; C12Q1/6813; C12Q1/6827; C12Q1/6834; C12Q1/6839; C12Q1/6844; C12Q1/6853; C12Q1/6855; C12Q1/6869; C12Q1/6874; C40; C40B; C40B80/00; G; G01; G01N; G01N37/00; B01J2219/00; B01J2219/00274; B01J2219/00277; B01J2219/00497; B01J2219/005; B01J2219/0054; B01J2219/00572; B01J2219/00583; B01J2219/00603; B01J2219/00605; B01J2219/0061; B01J2219/00612; B01J2219/00623; B01J2219/00626; B01J2219/0063; B01J2219/00632; B01J2219/00637; B01J2219/00646; B01J2219/00648; B01J2219/00659; B01J2219/00718; B01J2219/0072; B01J2219/00722; C40B20/00; C40B20/04; C40B40/00; C40B40/04; C40B40/06; C40B70/00
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
207 words
A method for determining the nucleotide sequence of a target polynucleotide, the method comprising the steps of: generating from the target polynucleotide a plurality of fragments that cover the target polynucleotide; attaching an oligonucleotide tag from a repertoire of tags to each fragment of the plurality (i) such that substantially all different fragments have different oligonucleotide tags attached and (ii) such that each oligonucleotide tag from the repertoire comprises a plurality of subunits and each subunit of the plurality consists of an oligonucleotide having a length from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set wherein a subunit of the set and a complement of any other subunit of the set would have at least two mismatches; sorting the fragments by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports and the respective complements being oligodeoxyribonucleotide N3'→P5' phosphoramidates or peptide nucleic acids; determining the nucleotide sequence of a portion of each of the fragments of the plurality; and determining the nucleotide sequence of the target polynucleotide by collating the sequences of the fragments.
Claim 2
Independent Claim
207 words
A method for determining the nucleotide sequence of a target polynucleotide, the method comprising the steps of: generating from the target polynucleotide a plurality of fragments that cover the target polynucleotide; attaching an oligonucleotide tag from a repertoire of tags to each fragment of the plurality (i) such that substantially all different fragments have different oligonucleotide tags attached and (ii) such that each oligonucleotide tag from the repertoire comprises a plurality of subunits and each subunit of the plurality consists of an oligonucleotide having a length from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set wherein a subunit of the set and a complement of any other subunit of the set would have at least two mismatches; sorting the fragments by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports and the respective complements being oligodeoxyribonucleotide N3'→P5' phosphoramidates or peptide nucleic acids; determining the nucleotide sequence of a portion of each of the fragments of the plurality; and determining the nucleotide sequence of the target polynucleotide by collating the sequences of the fragments.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein said oligonucleotide tags and said complements are single stranded oligonucleotides.
Claim 4
Dependent Claim
18 words
The method of claim 2 wherein said step of generating produces randomly overlapping fragments of said target polynucleotide.
Claim 5
Dependent Claim
31 words
The method of claim 3 wherein said step of determining said nucleotide sequence of said fragments is carried out simultaneously for said plurality of fragments by a single base sequencing method.
Claim 6
Dependent Claim
21 words
The method of claim 4 wherein each of said portions of said fragments is from 12 to 50 nucleotides in length.
Claim 7
Dependent Claim
21 words
The method of claim 5 wherein each of said portions of said fragments is from 12 to 25 nucleotides in length.
Claim 8
Dependent Claim
17 words
The method of claim 6 wherein said target polynucleotide is between one and fifty kilobases in length.
Claim 9
Dependent Claim
15 words
The method of claim 3 wherein said solid phase support is a plurality of microparticles.
Claim 10
Dependent Claim
21 words
The method of claim 8 wherein after said step of sorting, said plurality of microparticles is fixed to a planar substrate.
Claim 11
Dependent Claim
34 words
The method of claim 9 wherein said plurality of microparticles are disposed randomly on the surface of said planar substrate at a density of between 1000 microparticles to 100 thousand microparticles per square centimeter.
Claim 12
Dependent Claim
19 words
The method of claim 1 wherein said plurality of fragments is in the range of from 500 to 1000.
Claim 13
Independent Claim
136 words
A method for determining the nucleotide sequence of a target polynucleotide, the method comprising the steps of: generating from the target polynucleotide a plurality of fragments that cover the target polynucleotide; attaching an oligonucleotide tag from a repertoire of tags to each fragment of the plurality such that substantially all different fragments have different oligonucleotide tags attached; sorting the fragments by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports and the respective complements being oligodeoxyribonucleotide N3'→P5' phosphoramidates or peptide nucleic acids; determining the nucleotide sequence of a portion of each of the fragments of the plurality; and determining the nucleotide sequence of the target polynucleotide by collating the sequences of the fragments.
Claim 14
Independent Claim
136 words
A method for determining the nucleotide sequence of a target polynucleotide, the method comprising the steps of: generating from the target polynucleotide a plurality of fragments that cover the target polynucleotide; attaching an oligonucleotide tag from a repertoire of tags to each fragment of the plurality such that substantially all different fragments have different oligonucleotide tags attached; sorting the fragments by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports and the respective complements being oligodeoxyribonucleotide N3'→P5' phosphoramidates or peptide nucleic acids; determining the nucleotide sequence of a portion of each of the fragments of the plurality; and determining the nucleotide sequence of the target polynucleotide by collating the sequences of the fragments.
Claim 15
Dependent Claim
13 words
The method of claim 12 wherein said oligonucleotide tags are single stranded oligonucleotides.
Claim 16
Dependent Claim
18 words
The method of claim 13 wherein said step of generating produces randomly overlapping fragments of said target polynucleotide.
Claim 17
Dependent Claim
31 words
The method of claim 14 wherein said step of determining said nucleotide sequence of said fragments is carried out simultaneously for said plurality of fragments by a single base sequencing method.
Claim 18
Dependent Claim
21 words
The method of claim 15 wherein each of said portions of said fragments is from 12 to 50 nucleotides in length.
Claim 19
Dependent Claim
21 words
The method of claim 16 wherein each of said portions of said fragments is from 12 to 25 nucleotides in length.
Claim 20
Dependent Claim
17 words
The method of claim 17 wherein said target polynucleotide is between one and fifty kilobases in length.
Claim 21
Dependent Claim
18 words
The method of claim 12 wherein said one or more solid phase supports is a plurality of microparticles.
Claim 22
Dependent Claim
21 words
The method of claim 19 wherein after said step of sorting, said plurality of microparticles is fixed to a planar substrate.
Claim 23
Dependent Claim
34 words
The method of claim 20 wherein said plurality of microparticles are disposed randomly on the surface of said planar substrate at a density of between 1000 microparticles to 100 thousand microparticles per square centimeter.
Claim 24
Dependent Claim
23 words
The method of claim 12 wherein each of said oligonucleotide tags has a length in the range of from 10 to 40 monomers.
Claim 25
Dependent Claim
19 words
The method of claim 12 wherein said plurality of fragments is in the range of from 500 to 1000.
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| 3.5 Years (Year 4) | 2000-12-08 | 2001-06-09 | 2001-12-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2004-12-08 | 2005-06-09 | 2005-12-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2008-12-08 | 2009-06-09 | 2009-12-09 | $7700.00 | Window Closed / Lapsed |