Methods and kits for indirect labeling of nucleic acids

• US 7,371,519 B2
• Filed: 05/06/2003
• Issued: 05/13/2008
• Est. Priority Date: 01/31/2000
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Shannon, Karen W., Wolber, Paul K.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US10/431,335
• Publication Number:
  US 20040005609A1
• Time in Patent Office: 1,834 Days
• Field of Search: None
• US Class Current: 435/6.16
• 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 for comparing the expression profiles of at least two distinct samples, said method comprising:

(a) enzymatically generating;

(i) a first population of oligonucleotide tagged nucleic acids of differing sequence from an mRNA sample derived from a first sample, wherein each oligonucleotide tagged nucleic acid of said first population comprises a same first oligonucleotide tag; and

(ii) a second population of oligonucleotide tagged nucleic acids of differing sequence from an mRNA sample derived from a second sample, wherein each oligonucleotide tagged nucleic acid of said second population comprises a same second oligonucleotide tag that differs from said first oligonucleotide tag;

(b) hybridizing said first and second populations of oligonucleotide tagged target nucleic acids to an array of probe nucleic acids stably associated with the surface of a solid support, wherein said hybridizing occurs in the presence of;

(i) first labeled oligonucleotide complementary to said first oligonucleotide tag of said first population; and

(ii) second labeled oligonucleotide complementary to said second oligonucleotide tag of said second population;

whereby hybridized complexes comprising said oligonucleotide tagged target nucleic acids, probe nucleic acids and labeled oligonucleotides are produced on the surface of said array;

(c) detecting the presence of said hybridized complexes on said array surface;

(d) deriving an expression profile for each of said samples from said detected hybridized complexes; and

(e) comparing the derived expression profiles of each of said samples;

whereby the expression profiles of said at least two distinct samples are compared.

2. The method according to claim 1, wherein said first and second labels are distinguishable.

3. The method according to claim 2, wherein said first and second labels are fluorescent.

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

5. The method according to claim 4, wherein said primer further comprises an RNA polymerase promoter.

6. A method for producing a population of detectably labeled nucleic acids, said method comprising:

enzymatically generating from an mRNA source or sample by a linear amplification template dependent polymerization a population of oligonucleotide tagged nucleic acids of differing sequence using a primer comprising an oligonucleotide tag and template complementary region, wherein said oligonucleotide tagged nucleic acids in said population comprise the same oligonucleotide tag, and further wherein said oligonucleotide tagged nucleic acids in said population do not comprise a nucleotide residue modified to include a label moiety; and

contacting said oligonucleotide tagged nucleic acids with labeled oligonucleotides complementary to said oligonucleotide tag under conditions sufficient for said labeled oligonucleotide to hybridize to said oligonucleotide tag, wherein the length of the labeled oligonucleotide is substantially the same as that of the oligonucleotide tag;

whereby a population of detectably labeled nucleic acids is produced.

7. The method according to claim 6, wherein said detectably labeled nucleic acids are directly detectable.

8. The method according to claim 7, wherein said directly detectable label is fluorescent.

9. A method for producing a detectably labeled population of target nucleic acids from an initial nucleic acid sample, said method comprising:

enzymatically generating from an mRNA source or sample a population of oligonucleotide tagged target nucleic acids of differing sequence from an initial nucleic acid sample using a primer comprising an oligonucleotide tag and template complementary region, wherein said oligonucleotide tagged target nucleic acids in said population comprise the same oligonucleotide tag, and further wherein said oligonucleotide tagged nucleic acids in said population do not comprise a nucleotide residue modified to include a label moiety; and

contacting said population of oligonucleotide tagged target nucleic acids with labeled oligonucleotides complementary to said oligonucleotide tag of each oligonucleotide tagged target nucleic acid under hybridization conditions, wherein the length of the labeled oligonucleotide is substantially the same as that of the oligonucleotide tag;

whereby a detectably labeled population of target nucleic acids is produced.

10. The method according to claim 9, wherein said primer comprises an oligo dT domain.

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

12. The method according to claim 10, wherein said population of detectably labeled target nucleic acids is labeled with a fluorescent label.

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

(a) enzymatically generating from an mRNA source or sample by linear amplification a population of oligonucleotide tagged target nucleic acids of differing sequence from said nucleic acid analyte, wherein said oligonucleotide tagged target nucleic acids in said population comprise the same oligonucleotide tag, and further wherein said oligonucleotide tagged target nucleic acids in said population do not comprise a nucleotide residue modified to include a label moiety; and

(b) producing a three-part hybridized complex that comprises;

(i) an oligonucleotide tagged target nucleic acid from said population of oligonucleotide tagged target nucleic acids;

(ii) a labeled oligonucleotide hybridized to said oligonucleotide tag, wherein the length of the labeled oligonucleotide is substantially the same as that of the oligonucleotide tag; and

(iii) a probe nucleic acid, wherein said probe nucleic acid does not hybridize to the oligonucleotide tag of said oligonucleotide tagged target nucleic acid;

(c) detecting the presence of said hybridized complex; 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.

14. The method according to claim 13, wherein said probe is stably associated with the surface of a solid support.

15. The method according to claim 13, wherein said probe is present on an array.

16. The method according to claim 13, wherein said labeled oligonucleotide is fluorescently labeled.

17. The method according to claim 13, wherein a primer comprising an oligo dT region and said oligonucleotide tag is employed in said enzymatically generating step.

18. The method according to claim 17, wherein said primer further comprises an RNA polymerase promoter.

19. The method according to claim 13, wherein said method further comprises transmitting data obtained from at least one of said detecting and related steps to a remote location.

20. A method for obtaining an expression profile for a cell, said method comprising:

(a) enzymatically generating a population of oligonucleotide tagged nucleic acids of differing sequence from an mRNA sample derived from said cell, using a primer comprising an oligonucleotide tag and template complementary region, wherein each oligonucleotide tagged nucleic acid comprises a same oligonucleotide tag, and further wherein said oligonucleotide tagged nucleic acids in said population do not comprise a nucleotide residue modified to include a label moiety;

(b) producing at least one three-part hybridized complex comprising;

(i) an oligonucleotide tagged nucleic acid from said population of oligonucleotide tagged nucleic acids;

(ii) a labeled oligonucleotide hybridized to said oligonucleotide tag, wherein the length of the labeled oligonucleotide is substantially the same as that of the oligonucleotide tag; and

(iii) a probe nucleic acid stably associated with the surface of a solid support, wherein said probe nucleic acid does not hybridize to the oligonucleotide tag of said oligonucleotide tagged nucleic acid;

(c) detecting the presence of said at least one hybridized complex on said surface of said solid support; and

(d) deriving an expression profile for said cell from said detected at least one hybridized complex;

whereby said expression profile for said cell is obtained.

21. The method according to claim 20, wherein a primer comprising an oligo dT domain and said oligonucleotide tag is employed in said enzymatically generating step.

22. The method according to claim 21, wherein said primer further comprises an RNA polymerase promoter.

23. The method according to claim 20, wherein said labeled oligonucleotide is fluorescently labeled.

24. The method according to claim 20, wherein said method further comprises transmitting data obtained from at least one of said detecting and deriving steps to a remote location.