Methods and kits for indirect labeling of nucleic acids

• US 6,235,483 B1
• Filed: 01/31/2000
• Issued: 05/22/2001
• Est. Priority Date: 01/31/2000
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies Europe BV (Agilent Technologies, Inc.)
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Shannon, Karen W., Wolber, Paul K.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Wilder, Cynthia B.
• Application Number:
  US09/495,152
• Time in Patent Office: 477 Days
• Field of Search: 435/6, 435/810, 435/91.1, 435/325, 536/25.4, 536/24.2, 536/23.1, 536/24.33, 536/24.3, 935/77, 935/78, 365/45
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Methods and kits for labeling nucleic acids are provided. In the subject methods, an oligonucleotide tagged nucleic acid comprising an oligonucleotide tag is first generated. The oligonucleotide tagged nucleic acid is then contacted under hybridization conditions with a labeled oligonucleotide complementary to the oligonucleotide tag, yielding a labeled nucleic acid. The kits of the subject invention at least include a primer for use in enzymatically generating an oligonucleotide tagged target nucleic acid, where the primer generally at least includes an oligo dT region and the oligonucleotide tag, and a labeled oligonucleotide complementary to the oligonucleotide tag. The subject methods and kits find use in a variety of applications, and are particularly suited for use in gene expression analysis applications.

Claims

1. A method of detecting the presence of a nucleic acid analyte in a target sample, said method comprising:

(a) enzymatically generating an oligonucleotide tagged target nucleic acid from said nucleic acid analyte, wherein said oligonucleotide tagged target nucleic acid comprises an oligonucleotide tag; and

(b) producing a hybridized complex that comprises;

(i) said oligonucleotide tagged target nucleic acid;

(ii) a labeled oligonucleotide complementary to said oligonucleotide tag; and

(iii) a probe nucleic acid, wherein said labeled oligonucleotide is hybridized to said oligonucleotide tag and said probe nucleic acid is hybridized to a non-tag region of said oligonucleotide tagged target nucleic acid;

(c) detecting the presence of said hybridized complex by detecting the presence of said labeled oligonucleotide; and

(d) relating the presence of said hybridized complex to the presence of said nucleic acid analyte in said sample;

whereby the presence of said nucleic acid analyte in said sample is detected.

2. The method according to claim 1, wherein said probe is stably associated with the surface of a solid support.

3. The method according to claim 1, wherein said probe is present on an array.

4. The method according to claim 1, wherein said labeled oligonucleotide is fluorescently labeled.

5. The method according to claim 1, wherein a primer comprising an oligo dT region and said oligonucleotide tag is employed in said enzymatically generating step.

6. The method according to claim 5, wherein said primer further comprises an RNA polymerase promoter.

7. The method according to claim 1, wherein said method further comprises transmitting data obtained from at least one of said detecting and relating steps to a remote location.

8. A method for obtaining an expression profile for at least a representative number of genes in a cell, said method comprising:

(a) enzymatically generating a population of oligonucleotide tagged nucleic acids from an mRNA sample derived from said cell, wherein each oligonucleotide tagged nucleic acid comprises an oligonucleotide tag;

(b) producing hybridized complexes each comprising;

(i) an oligonucleotide tagged target nucleic acid;

(ii) a labeled oligonucleotide complementary to said oligonucleotide tag; and

(iii) a probe nucleic acid stably associated with the surface of a solid support, wherein said labeled oligonucleotide is hybridized to said oligonucleotide tag and said probe nucleic acid is hybridized to a non-tag region of said oligonucleotide tagged target nucleic acid;

(c) detecting the presence of said hybridized complexes on said array surface by detecting the presence of said labeled oligonucleotide; and

(d) obtaining an expression profile for said cell from said detected hybridized complexes;

whereby said expression profile for at least a representative number of genes in said cell is obtained.

9. The method according to claim 8, wherein a primer comprising an oligo dT domain and said oligonucleotide tag is employed in said enzymatically generating step.

10. The method according to claim 9, wherein said primer further comprises an RNA polymerase promoter.

11. The method according to claim 8, wherein said labeled oligonucleotide is fluorescently labeled.

12. The method according to claim 8, wherein said method further comprises transmitting data obtained from at least one of said detecting and deriving steps to a remote location.