METHODS OF ENZYMATIC DISCRIMINATION ENHANCEMENT AND SURFACE-BOUND DOUBLE-STRANDED DNA

• US 20100216656A1
• Filed: 08/07/2008
• Published: 08/26/2010
• Est. Priority Date: 10/21/1994
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Vetter, Dirk, Digglemann, Martin, Chee, Mark S., Lockhart, David J.
• Granted Patent: US 8,236,493 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01J 2219/00427 using masks
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• CPC Class Codes: C40B 60/12 for screening libraries

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.

Claims

1. A method for identifying a base, the method comprising:

a) hybridizing;

a substrate comprising an array of oligonucleotides each of which is complementary to a defined subsequence of preselected length, and

a target nucleic acid, thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence;

b) hybridizing the target-oligonucleotide probe hybrid complexes with a pool of labeled, ligatable oligonucleotide probes of a preselected length,

c) ligating the labeled ligatable oligonucleotide to the target-oligonucleotide hybrid complex with a ligase; and

d) identifying which base has specifically interacted with the target nucleic acid as an indication of a subsequence that is complementary.

2. The method according to claim 1 wherein the target nucleic acid is ribonucleic acid (RNA).

3. The method according to claim 1 wherein the target nucleic acid is deoxyribonucleic acid (DNA).

4. The method of claim 1, wherein the array of target;

oligonucleotide hybrid complexes has been formed on a substrate comprising beads.

5. A method for analyzing a target nucleic acid, the method comprising:

a) providing a reaction system comprising;

a heating mechanism

at least one cavity next to the heating mechanism and

a fluorescence detector next to the cavity

b) providing a reaction chamber comprising

a substrate comprising an array of oligonucleotides each of which is complementary to a defined subsequence of preselected length

a target nucleic acid

c) hybridizing the target nucleic acid to at least one of the oligonucleotides to form target-oligonucleotide hybrid complexes of complementary subsequences of known sequence using the heating mechanism;

d) contacting the target-oligonucleotide probe hybrid complexes with a ligase and a pool of labeled, ligatable oligonucleotide probes of a preselected length,

e) positioning the reaction chamber to the cavity such that substrate surface faces the fluorescence detector; and

f) determining which of the oligonucleotides contain the labeled, ligatable oligonucleotide probe as an indication of a subsequence which is complementary to a subsequence of the target nucleic acid.

6. A diagnostic kit comprising:

a) a heating mechanism;

b) at least one cavity next to the heating mechanism;

c) a fluorescence detector next to the cavity

d) a reaction chamber;

e) at least one substrate comprising an array of oligonucleotide probes of a preselected length;

f) a target oligonucleotide to be sequenced;

g) a pool of labeled, ligatable oligonucleotide probes of a preselected length; and

h) a ligase, thereby forming target-oligonucleotide hybrid complexes of complementary subsequences of known sequence.

a) A data analysis method for analyzing target oligonucleotide sequencing data.

7-36. -36. (canceled)