Patent EP0721016A2 successfully refreshed from Google Patents.
EP0721016A2
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Nucleic acid library arrays, methods for synthesizing them and methods for sequencing and sample screening using them
Assignee: Affymax Technologies NV
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Filing: 1995-10-20
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Granted: —
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Expires: 2015-10-20
Abstract
Abstract Methods for discriminating between fully complementary hybrids and those that differ by one or more base pairs and libraries of unimolecular, double-stranded oligonucleotides on a solid support. In one embodiment, the present invention provides methods of using nuclease treatment to improve the quality of hybridization signals on high density oligonucleotide arrays. In another embodiment, the present invention provides methods of using ligation reactions to improve the quality of hybridization signals on high density oligonucleotide arrays. In yet another embodiment, the present invention provides libraries of unimolecular or intermolecular, double-stranded oligonucleotides on a solid support. These libraries are useful in pharmaceutical discovery for the screening of numerous biological samples for specific interactions between the double-stranded oligonucleotides, and peptides, proteins, drugs and RNA. In a related aspect, the present invention provides libraries of conformationally restricted probes on a solid support. The probes are restricted in their movement and flexibility using double-stranded oligonucleotides as scaffolding. The probes are also useful in various screening procedures associated with drug discovery and diagnosis. The present invention further provides methods for the preparation and screening of the above libraries.
Inventors
- 👤 David J. Lockhart
- 👤 Mark S. Chee
- 👤 Dirk Vetter
- 👤 Martin Diggelmann
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 21:22
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1093; B; B01; B01J; B01J19/00; B01J19/0046; C07; C07H; C07H21/00; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q1/6834; C12Q1/6837; C12Q1/6869; C12Q1/6874; C40; C40B; C40B80/00; B01J2219/00; B01J2219/00274; B01J2219/00277; B01J2219/00497; B01J2219/00527; B01J2219/00529; B01J2219/00583; B01J2219/00603; B01J2219/00605; B01J2219/00608; B01J2219/00612; B01J2219/00623; B01J2219/00626; B01J2219/0063; B01J2219/00659; B01J2219/00718; B01J2219/0072; B01J2219/00722; C07B; C07B2200/00; C07B2200/11; C40B40/00; C40B40/04; C40B40/06; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/5308; G01N33/543; G01N33/551
Claims Analysis
Total Claims: 33
Claim 1
Independent Claim
99 words
A method for sequencing a target nucleic acid, said method comprising: (a) combining: (i) a substrate comprising an array of chemically synthesized and positionally distinguishable oligonucleotides each of which is complementary to a defined subsequence of preselected length; and (ii) a target nucleic acid; thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence; (b) contacting said target-oligonucleotide hybrid complexes with a nuclease; thereby removing target-oligonucleotide complexes which are not perfectly complementary; and (c) determining which of said oligonucleotides have specifically interacted with subsequences in said target nucleic acid, to determine the sequence of said target nucleic acid.
Claim 2
Dependent Claim
26 words
The method as recited in claim 1 wherein said target nucleic acid is ribonucleic acid (RNA), optionally said nuclease is an RNA nuclease, preferably RNase A.
Claim 3
Independent Claim
124 words
A method for sequencing a target nucleic acid, said method comprising: (a) combining: (i) a substrate comprising an array of chemically synthesized and positionally distinguishable oligonucleotides each of which is complementary to a defined subsequence of preselected length; and (ii) a target nucleic acid which is longer than each of said probes; thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence with a 3' target overhang; (b) contacting said target-oligonucleotide hybrid complexes with a ligase and a labelled, ligatable oligonucleotide probe; (c) removing unbound target nucleic acid and labelled, unligated oligonucleotide probes; and (d) determining which of said oligonucleotides contain said labelled, ligatable oligonucleotide probe as an indication of a subsequence which is complementary to a subsequence of said target nucleic acid.
Claim 4
Dependent Claim
19 words
The method as recited in claim 1 or claim 3 wherein said target nucleic acid is deoxyribonucleic acid (DNA).
Claim 5
Dependent Claim
28 words
The method as recited in claim 4 when dependent on claim 1 wherein said nuclease is a DNA nuclease, preferably DNA nuclease S1 nuclease or Mung Bean nuclease.
Claim 6
Independent Claim
27 words
The method as recited in any preceding claim wherein said array of oligonucleotides recognizes substantially all possible subsequences of preselected length found in said target nucleic acid.
Claim 7
Independent Claim
28 words
The method as recited in any preceding claim, wherein each oligonucleotide is of a length between about 6 and 20 bases, preferably between about 8 and 15 bases.
Claim 8
Independent Claim
44 words
The method as recited in any preceding claim, wherein said array of oligonucleotides comprises about 1,000 different oligonucleotides, preferably about 3,000 different oligonucleotides, preferably about 10 4 different oligonucleotides, more preferably about 10 5 different oligonucleotides, even more preferably about 10 6 different oligonucleotides.
Claim 9
Independent Claim
40 words
The method as recited in any one of claims 3, 4 or 6 to 8 wherein said ligase is a member selected from the group consisting of T4 DNA ligase, ligases isolated from E. coli and ligases isolated from bacteriophages.
Claim 10
Independent Claim
166 words
A method for sequencing an unlabeled target oligonucleotide, said method comprising: (a) combining: (i) a substrate comprising an array of positionally distinguishable oligonucleotide probes each of which has a constant region and a variable region, said variable region capable of binding to a defined subsequence of preselected length; (ii) a constant oligonucleotide having a sequence which is complementary to said constant region of said oligonucleotide probes; (iii)a target oligonucleotide to be sequenced; and (iv) a ligase, thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence; (b) contacting said target oligonucleotide-oligonucleotide probe hybrid complexes with a ligase and a pool of labelled, ligatable oligonucleotide probes of a preselected length, said pool of labelled, ligatable oligonucleotide probes representing all possible sequences of said preselected length; (c) removing unbound target nucleic acid and labelled, unligated oligonucleotide probes; and (d) determining which of said oligonucleotide probes contain said labelled, ligatable oligonucleotide probe as an indication of a subsequence which is complementary to a subsequence of said target oligonucleotide.
Claim 11
Independent Claim
121 words
A method for sequencing an unlabelled target oligonucleotide, said method comprising; (a) contacting an unlabelled target oligonucleotide with a library of labelled oligonucleotide probes, each of said oligonucleotide probes having a known sequence and being attached to a solid support at a known position, to hybridize said target oligonucleotide to at least one member of said library of probes, thereby forming a hybridized library; (b) contacting said hybridized library with a nuclease capable of cleaving double-stranded oligonucleotides to release from said hybridized library a portion of said labelled oligonucleotide probes or fragments thereof; and (c) identifying said positions of said hybridized library from which labelled probes or fragments thereof have been removed, to determine the sequence of said unlabelled target oligonucleotide.
Claim 12
Dependent Claim
67 words
A synthetic unimolecular, double-stranded oligonucleotide library comprising a plurality of different members, each member having the formula: Y-L 1 -X 1 -L 2 -X 2 wherein, Y is a solid support; X 1 and X 2 are a pair of complementary oligonucleotides L 1 is a spacer; L 2 is a linking group having sufficient length such that X 1 and X 2 form a double-stranded oligonucleotide.
Claim 13
Dependent Claim
35 words
A library in accordance with claim 12, wherein L 2 is a member selected from the group consisting of an alkylene group, a polyethyleneglycol group, a polyalcohol group, a polymine group and a polyester group.
Claim 14
Dependent Claim
29 words
A library in accordance with claim 12 or claim 13, wherein X 1 and X 2 are complementary oligonucleotides each comprising of from 6 to 30 nucleic acid monomers.
Claim 15
Dependent Claim
32 words
A library in accordance with any one of claims 12 to 14, wherein said solid support is a silica support and L 1 comprises an aminoalkylsilane and from 1 to 4 hexaethyleneglycols.
Claim 16
Dependent Claim
35 words
A synthetic unimolecular, double-stranded oligonucleotide library of any one of claims 12 to 15, wherein a portion of said double-stranded oligonucleotides formed by X 1 and X 2 further comprise a bulge or a loop.
Claim 17
Dependent Claim
35 words
A synthetic unimolecular, double-stranded nucleic acid library of any one of claims 12 to 16, wherein each member further comprises an identifier tag, said identifier tag identifying the sequence of said unimolecular, double-stranded nucleic acid.
Claim 18
Dependent Claim
49 words
A synthetic unimolecular, double-stranded nucleic acid library of any one of claims 12 to 17, wherein said solid support comprises a first bead linked to a second bead, wherein the double-stranded nucleic acid is attached to the first bead and an identifier tag is attached to the second bead.
Claim 19
Independent Claim
130 words
A method of forming a plurality of diverse unimolecular, double-stranded oligonucleotides on a solid support having optional spacers, said support comprising a surface with a plurality of preselected regions, said method comprising: (a) forming on each of said preselected regions a different first oligonucleotide, each of said first oligonucleotides comprising of from 6 to 30 monomers; (b) attaching to the distal end of each of said first oligonucleotides of step (a) a linking group; and (c) forming on the distal end of each of said linking groups a second oligonucleotide, wherein each of said second oligonucleotides is complementary to said first oligonucleotide which is attached within the same preselected region, and wherein said linking groups have sufficient length such that said first and second oligonucleotides form a unimolecular, double-stranded oligonucleotide.
Claim 20
Independent Claim
96 words
A method of screening a sample for a species capable of binding to double-stranded DNA comprising: contacting said sample with a solid support comprising unimolecular, double-stranded DNA attached thereon, each of said attached DNA independently having the formula; -X 11 -L-X 12 wherein, X 11 and X 12 are complementary oligonucleotides; and L is a linking group having sufficient length such that X 11 and X 12 form said attached unimolecular, double-stranded DNA, to produce at least one bound pair comprising said species and one of said attached unimolecular, double-stranded DNA; and identifying said bound pair.
Claim 21
Dependent Claim
27 words
A method in accordance with claim 20, wherein said species is a member selected from the group consisting of a drug, a protein and an RNA molecule.
Claim 22
Independent Claim
90 words
A method of screening a sample for a species capable of binding to double-stranded DNA comprising: contacting said sample with a solid support comprising a unimolecular, double-stranded DNA attached thereon, said attached DNA having the formula; -X 11 -L-X 12 wherein, X 11 and X 12 are complementary oligonucleotides; and L is a linking group having sufficient length such that X 11 and X 12 form said attached unimolecular, double-stranded DNA, to produce a bound pair comprising said species and said attached unimolecular, double-stranded DNA; and identifying said bound pair.
Claim 23
Dependent Claim
69 words
A synthetic conformationally-restricted probe library comprising a plurality of members, each of said members comprising a solid support attached to an oligomer having the formula: -X 11 -Z-X 12 wherein, X 11 and X 12 are complementary oligonucleotides; and Z is a probe having sufficient length such that X 11 and X 12 form a double-stranded portion of said member and thereby restrict the conformations available to said probe.
Claim 24
Dependent Claim
35 words
A synthetic library in accordance with claim 23, wherein each of said probes is a peptide having of from about 4 to about 12 amino acids and optionally each member further comprises an intercalating dye.
Claim 25
Independent Claim
128 words
A method of synthesizing a library of conformationally-restricted probes on a solid support having optional spacers, said support comprising a surface with a plurality of preselected regions, said method comprising: (a) forming on each of said preselected regions a first oligonucleotide, each of said first oligonucleotides comprising of from 6 to 30 monomers; (b) attaching to the distal end of each of said first oligonucleotides of step (a) a probe; and (c) forming on the distal end of each of said probes a second oligonucleotide, wherein each of said second oligonucleotides is complementary to said first oligonucleotide which is attached within the same preselected region, and wherein said probes have sufficient length such that said first and second oligonucleotides form a unimolecular, double-stranded oligonucleotide thereby conformationally-restricting said probes.
Claim 26
Dependent Claim
25 words
A method in accordance with claim 19 or claim 25, wherein said method of construction of step (a) and step (b) is by light-directed synthesis.
Claim 27
Independent Claim
97 words
A method of screening a sample for a species capable of binding to a conformationally-restricted probe comprising: contacting said sample with a solid support comprising conformationally-restricted probes attached thereon, each of said attached probes independently having the formula; -X 11 -Z-X 12 wherein, X 11 and X 12 are complementary oligonucleotides; and Z is a probe having sufficient length such that X 11 and X 12 form a double-stranded oligonucleotide portion of said conformationally-restricted probe, to produce at least one bound pair comprising said species and one of said attached conformationally-restricted probes; and identifying said bound pair.
Claim 28
Dependent Claim
44 words
An adhesive for use in biological applications comprising a first surface having a plurality of attached oligonucleotides and a second surface having a plurality of attached oligonucleotides, wherein the oligonucleotides of said first surface are substantially complementary to the oligonucleotides of said second surface.
Claim 29
Dependent Claim
48 words
A synthetic intermolecular, doubly-anchored, double-stranded oligonucleotide library comprising a plurality of different members, each member having the formula: wherein, Y is a solid support; X 1 and X 2 are a pair of complementary oligonucleotides; L 1 and L 2 are each independently a bond or a spacer.
Claim 30
Dependent Claim
53 words
A library in accordance with claim 29, wherein L 1 and L 2 each independently comprise a member selected from the group consisting of an alkylene group, a polyethyleneglycol group, a polyalcohol group, a polyamine group and a polyester group, preferably L 1 and L 2 each independently comprise a polyethylene glycol group.
Claim 31
Dependent Claim
29 words
A library in accordance with claim 29 or claim 30, wherein X 1 and X 2 are complementary oligonucleotides each comprising of from 6 to 30 nucleic acid monomers.
Claim 32
Dependent Claim
32 words
A library in accordance with any one of claims 29 to 31, wherein said solid support is a silica support and L 1 comprises an aminoalkylsilane and from 1 to 4 hexaethyleneglycols.
Claim 33
Independent Claim
80 words
A method of preparing a single-stranded nucleic acid sequence, said method comprising: (a) forming a hybrid complex by combining at least two oligonucleotides which are phosphorylated at their 5' ends with a chip-bound oligonucleotide, said chip-bound oligonucleotide having subsequences which are complementary to a subsequence of each of said oligonucleotide; (b) contacting said hybrid complex with a ligase to form a ligated oligonucleotide; and (c) releasing said ligated oligonucleotide from said chip-bound oligonucleotide to form a single-stranded nucleic acid sequence.
References Cited (Backward Citations)
No citation records indexed.
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