Methods of labeling nucleic acids for use in array based hybridization assays

• US 6,383,749 B2
• Filed: 12/02/1999
• Issued: 05/07/2002
• Est. Priority Date: 12/02/1999
• Status: Expired due to Term

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Clontech Laboratories Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Bochkariov, Dmitry E., Chenchik, Alex
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/454,183
• Publication Number:
  US 20010007747A1
• Time in Patent Office: 887 Days
• Field of Search: 435/6, 435/91.2, 435/320.1, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

Methods and kits are provided for labeling nucleic acids, e.g. for use in array based hybridization assays. In the subject methods, target nucleic acid is generated from an initial nucleic acid source, e.g. mRNA, where the target nucleic acid is characterized by having at least one reactive functionality that is not a moiety found on naturally occurring nucleic acids. Functionalized label is then conjugated to the target nucleic acid, either before or after it has been hybridized to array of nucleic acids stably associated with the surface of a solid support. The subject methods find use in a variety of array based hybridization assays, including differential expression assays.

Claims

1. A method of producing a population of labeled target nucleic acids, said method comprising:

generating a population of target nucleic acids comprising a first reactive functionality, wherein said first reactive functionality is a moiety that is not present on naturally occurring nucleic acids and said population of target nucleic acids is generated by contacting an initial mRNA sample with a fluid composition of at least 50 distinct gene specific primers under conditions sufficient to enzymatically generate said population of target nucleic acids, wherein each constituent distinct gene specific primer of said fluid composition has a nucleotide sequence that is complementary to a distinct mRNA and wherein each target nucleic acid is generated using a single gene specific primer; and

contacting said population of nucleic acids with a functionalized label comprising a second reactive functionality under conditions sufficient for said first and second reactive functionalities to react with each other to produce a covalent bond between said label and said nucleic acid to produce said population of labeled target nucleic acids.

2. The method according to claim 1, wherein said label is a directly detectable label.

3. The method according to claim 2, wherein said label is a fluorescent label.

4. The method according to claim 1, wherein said population of target nucleic acids is enzymatically generated.

5. The method according to claim 4, wherein at least one nucleotide functionalized with said first functionality is employed in said generating step.

6. A method of hybridizing a population of target nucleic acids to an array made up of a plurality of probe nucleic acids stably associated with the surface of a solid support, said method comprising:

generating said population of target nucleic acids by contacting an initial mRNA sample with a fluid composition of at least 50 distinct gene specific primers under conditions sufficient to enzymatically generate said population of target nucleic acids, wherein each constituent distinct gene specific primer of said fluid composition has a nucleotide sequence that is complementary to a distinct mRNA and wherein each target nucleic acid is generated using a single gene specific primer, wherein each constituent target nucleic acid comprises a first reactive functionality, wherein said first reactive functionality is a moiety that is not present on naturally occurring nucleic acids; and

hybridizing said generated population of target nucleic acids to said array;

with the proviso that said method further comprises a step of conjugating a functionalized label comprising a second reactive functionality to said population of target nucleic acids by a reaction between said first and second reactive functionalities.

7. The method according to claim 6, wherein said conjugating occurs prior to said hybridizing.

8. The method according to claim 6, wherein said conjugating occurs after said hybridizing.

9. The method according to claim 6, wherein said first reactive functionality is selected from the group consisting of amino, sulfhydryl, azido, isothiocyanate and sulfoxyl.

10. The method according to claim 6, wherein said second reactive functionality is an N-hydroxysuccinimide ester moiety.

11. The method according to claim 6, wherein said label is directly detectable.

12. The method according to claim 11, wherein said label is a fluorescent label.

13. The method according to claim 6, wherein said population of target nucleic acids is enzymatically generated.

18. The array produced according to claim 6.

14. An array based hybridization method, said method comprising:

enzymatically generating a plurality of amino modified distinct target nucleic acids from an initial RNA source by contacting said initial mRNA source with a fluid composition of at least 50 distinct gene specific primers under conditions sufficient to enzymatically generate said population of target nucleic acids, wherein each constituent distinct gene specific primer of said fluid composition has a nucleotide sequence that is complementary to a distinct mRNA and wherein each target nucleic acid is generated using a single gene specific primer;

contacting N-hydroxysuccinimide ester functionalized label with said plurality of amino modified distinct target nucleic acids under conditions sufficient to produce a plurality of labeled target nucleic acid conjugates; and

hybridizing said plurality of labeled target nucleic acid conjugates to an array of a plurality of distinct nucleic acid probes stably associated with the surface of a solid support.

15. The method according to claim 14, wherein said label is a fluorescent label.

16. An array based hybridization method, said method comprising:

(a) enzymatically generating a plurality of amino modified distinct target nucleic acids from an initial nucleic acid source by contacting an initial mRNA sample with a fluid composition of at least 50 distinct gene specific primers under conditions sufficient to enzymatically generate said population of target nucleic acids, wherein each constituent distinct gene specific primer of said fluid composition has a nucleotide sequence that is complementary to a distinct mRNA and wherein each target nucleic acid is generated using a single gene specific primer;

(b) hybridizing said plurality of amino modified target nucleic acid conjugates to an array of a plurality of distinct nucleic acid probes stably associated with the surface of a solid support to produce at least one duplex of an amino modified target nucleic acid and a probe nucleic acid on said surface of said array; and

(c) contacting N-hydroxysuccinimide ester functionalized label with said surface of said array under conditions sufficient for said label to conjugate to said amino modified target nucleic acid.

17. The method according to claim 16, wherein said label is a fluorescent label.

19. A kit for use in labeling a nucleic acid, said kit comprising:

(a) nucleotides;

(b) functionalized nucleotide analogs comprising a first reactive functionality not present in naturally occurring nucleic acids;

(c) functionalized label comprising a second reactive functionality, wherein said second reactive functionality is capable of reacting with said first reactive functionality; and

(d) a fluid composition of at least 50 distinct gene specific primers wherein each constituent distinct gene specific primer of said fluid composition has a nucleotide sequence that is complementary to a distinct mRNA.

20. The kit according to claim 19, wherein said kit further comprises an array of a plurality of probes stably associated with the surface of a solid support.