Methods of enzymatic discrimination enhancement and surface-bound double-stranded DNA

• US 8,236,493 B2
• Filed: 08/07/2008
• Issued: 08/07/2012
• Est. Priority Date: 10/21/1994
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Lockhart, David J., Chee, Mark S., Vetter, Dirk, Digglemann, Martin
• Primary Examiner(s): Babic, Christopher M.
• Application Number:
  US12/187,503
• Publication Number:
  US 20100216656A1
• Time in Patent Office: 1,461 Days
• Field of Search: None
• US Class Current: 435/6
• 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 at least 10⁶ different oligonucleotides wherein the oligonucleotides are attached to the substrate via the 3′

ends of the oligonucleotides, the 5′

ends of the oligonucleotides are phosphorylated, each different oligonucleotide is present in a different predefined region of the array, and each of the different oligonucleotides is complementary to a defined subsequence of a target nucleic acid, and

a nucleic acid sample comprising target nucleic acids, wherein the target nucleic acids hybridize to their complementary oligonucleotides leaving a single stranded 3′

overhanging region of the hybridized target nucleic acid, thereby forming target-oligonucleotide hybrid complexes having a single stranded 3′

overhanging region;

b) hybridizing the target-oligonucleotide probe hybrid complexes with a pool of labeled, ligatable oligonucleotide probes of a preselected length to form complexes comprising labeled, ligatable probes hybridized to the 3′

overhanging region of the hybridized target nucleic acid adjacent to the 5′

end of the oligonucleotides;

c) ligating the labeled ligatable oligonucleotide probes to the 5′

end of the oligonucleotides with a ligase thereby forming labeled oligonucleotides on the substrate;

d) washing the substrate to remove target nucleic acids and unligated labeled, ligatable probes; and

e) detecting the presence of labeled oligonucleotides on the substrate to identify 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. The method of claim 1 wherein the 5′

ends of the oligonucleotides are phosphorylated by treating the array with kinase prior to the hybridization of the labeled, ligatable probes.