Hybridization of target DNA with immobilized nucleic acid analogs

• US 7,270,954 B1
• Filed: 06/30/2000
• Issued: 09/18/2007
• Est. Priority Date: 06/30/2000
• Status: Expired due to Fees

• Current Assignee: Iris Technologies, Inc.
• Original Assignee: Iris Technologies, Inc.
• Inventors: Ramirez-Vick, Jaime E.
• Primary Examiner(s): Lam; Ann Yen
• Application Number:
  US10/333,697
• Time in Patent Office: 2,636 Days
• Field of Search: 435/6, 435/7, 435/94, 435/518, 435/4, 435/7.1, 435 74- 795, 4352831-2891, 435/973, 536/24.3, 536/25.3, 436/514, 436523-528, 422 50- 73
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

This invention is related to the immobilization of peptide nucleic acids (PNAs) onto solid surfaces for use in hybridization, purification, biosensing, and other biochemical applications. Specifically, their use to increase the thermal stability, specificity, and lifetime of devices based on in situ hybridization is disclosed. A method is disclosed by which peptide nucleic acids replace oligonucleotides as probes in the detection of specific DNA and RNA sequences.

Claims

1. A microarray comprising a solid support surface comprising a plurality of reusable ligand-binding specific reaction sites comprising:

a) a soft metal solid support,

b) a heterobifunctional spacer comprising a flexible spacer arm and chemi- or physisorbed to said soft metal solid support via soft metal-soft base bonding, wherein said heterobifunctional spacer comprises

1) an anchor group comprising a soft base selected from the group consisting of succinimidyl-6-(biotinamido)hexanoate and dithiobis(succinimidyl-undecanoate), and

2) a reactive terminal group selected from the group consisting of an isothiocyanate and an isocyanate, and

c) a peptide nucleic acid (PNA) linked to said reactive terminal group, wherein said flexible spacer arm allows the PNA to interact with its environment in a way which minimizes any steric hindrance,

said specific reaction site surfaces comprising PNA solutions of different compositions at different sites on the solid support surface.

2. A microarray comprising a solid support surface of claim 1 in which the soft metal solid support is selected from the group consisting of silver, copper, gold, platinum (II), mercury, mercury (II), thallium, cadmium (II), platinum (IV) and palladium (II) covered surfaces.

3. A microarray comprising a solid support surface of claim 1 in which the heterobifunctional spacer does not contain heteroatoms.

4. A microarray comprising a solid support surface of claim 1 wherein the soft base is selected from the group consisting of an RSH, RS⁻

, R₂S, RSSR, CN⁻

, S₂O₃²⁻

, I⁻

, R₃P, (RO)₃P, C₂H₄ and C₆H₆ group, where R is an organic group.

5. The microarray comprising a solid support surface of claim 1, wherein said specific reaction sites are disposed at a surface density greater than about 100 specific reaction sites per square centimeter and detecting the presence of the biological molecule.

6. A microarray comprising a solid support surface of claim 1, wherein said reactive terminal group is an isocyanate reactive terminal group.

11. A method for detecting the presence of a biological molecule comprising exposing a sample containing biological molecules to a solid support surface of claim 1.

7. A method for preparing a microarray comprising a solid support surface comprising a plurality of reusable ligand-binding specific reaction sites comprising:

a) selecting a soft metal solid support,

b) immobilizing at each specific reaction site a heterobifunctional spacer comprising a flexible spacer arm on said solid support via soft metal-soft base bonding, wherein said heterobifunctional spacer comprises

1) an anchor group comprising a soft base selected from the group consisting of succinimidyl-6-(biotinamido)hexanoate and dithiobis(succinimidyl-undecanoate), and

2) a reactive terminal group selected from the group consisting of an isothiocyanate, an isocyanate, and an N-hydroxysuccinimide ester, and

c) attaching at each specific reaction site a PNA to said reactive terminal group of said spacer by addition of PNA in an aqueous solution at a molar ratio of spacer;

PNA of between about 2;

1 to 50;

1, wherein said flexible spacer arm allows the PNA to interact with its environment in a way which minimizes any steric hindrance,

said specific reaction site surfaces comprising PNA solutions of different compositions at different sites on the solid support surface.

8. A method of claim 7 in which the soft metal solid support is selected from the group consisting of silver, copper, gold, platinum (II), mercury, mercury (II), thallium, cadmium (II), platinum (IV) and palladium (II) covered surfaces.

9. A method of claim 7 in which the heterobifunctional spacer does not contain heteroatoms.

10. A method of claim 7 wherein the soft base is selected from the group consisting of an RSH, RS⁻

, R₂S, RSSR, CN⁻

, S₂O₃²⁻

, I⁻

, R₃P, (RO)₃P, C₂H₄ and C₆H₆ group, where R is an organic group.

12. The method of claim 7 for preparing a microarray comprising a solid support surface, wherein said immobilizing step comprises selecting a reactive terminal group from the group consisting of an isothiocyanate and an isocyanate.

13. The method of claim 7 for preparing a microarray comprising a solid support surface, wherein said immobilizing step comprises selecting an isocyanate for said reactive terminal group.

14. The method of claim 7, wherein said specific reaction sites are disposed at a surface density greater than about 100 specific reaction sites per square centimeter.