Derivative nucleic acids and uses thereof

• US 6,964,847 B1
• Filed: 07/14/2000
• Issued: 11/15/2005
• Est. Priority Date: 07/14/1999
• Status: Expired due to Term

• Current Assignee: Packard Biosciences, Inc.
• Original Assignee: Packard Bioscience Co. (Massachusetts) (Revvity, Inc.)
• Inventors: Englert, David F.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Assistant Examiner(s): Chakrabarti, Arun
• Application Number:
  US09/616,787
• Time in Patent Office: 1,950 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3, 536/23.1, 536/32, 536/33, 935/6, 436/518
• US Class Current: 435/6.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The invention features a method for multiplexed analysis of a plurality of target nucleic acid sequences in a sample. The method provides a derivative nucleic acid for each target sequence analyzed and present in the sample.

Claims

1. A method of analyzing a sample of nucleic acids, the method comprising:

providing a plurality of probe molecules wherein the plurality comprises at least one probe molecule for each target nucleic acid sequence to be analyzed, the probe molecule comprising a region of sequence substantially complementary to a sequence in the target nucleic acid sequence and an internal capture tag sequence that is internal to a nucleic acid strand of said probe molecule;

contacting the plurality of probe molecules to a sample of nucleic acids under conditions that allow a set of probe molecules for which a complementary sequence is present among the nucleic acids of the sample, to hybridize to the respective complementary sequence;

cleaving the probes molecules of the set, wherein the cleavage is specific for the probe molecules that hybridize to nucleic acids of the sample and the cleavage positions the capture tag sequence of each cleaved probe molecule at a terminus of the cleaved probe molecule; and

detecting one or more of the cleaved probes by hybridizing the tag sequences to capture probes disposed on a substrate, thereby analyzing the sample of nucleic acids.

2. The method of claim 1 herein each probe molecule comprises a Type IIS restriction endonuclease recognition site positioned such that cleavage of the recognition site in a double studded DNA into which the probe molecule is incorporated generates a nucleic acid having a single-stranded overhang that includes the tag sequence.

3. The method of claim 1, wherein the cleaving positions the capture tag sequence of each cleaved probe molecule in a single-stranded overhang.

4. The method of claim 3, wherein detecting comprises hybridizing capture tag sequences of the one or more cleaved probes to a plurality of capture probes.

5. The method of claim 4, where in each capture probe of the plurality of capture probes comprises a double stranded region and a single stranded region.

6. The method of claim 5, wherein the 3′

end of the single stranded region is extendable.

7. The method of claim 5, wherein each capture probe of the plurality of capture probes forms a hairpin structure.

8. The method of claim 5, wherein each capture probe of the plurality of capture probes comprises a chemical moiety that allows for immobilization.

9. The method of claim 1 wherein detecting comprises hybridizing capture tag sequences of the one or more cleaved probes to a plurality of capture probes.

10. The method of claim 9, wherein the detecting comprises an enzyme mediated reaction.

12. The method of claim 10 wherein a capture probe is a substrate or template for the enzyme mediated reaction.

13. The method of claim 1 wherein the internal capture tag sequence of each probe molecule of the plurality of molecules is between 4 and 20 nucleotides in length.

14. The method of claim 1 wherein the internal capture tag sequence of each probe molecule of the plurality of probe molecules is 4 and 8 nucleotides in length.

15. The method of claim 1 wherein the internal capture tag sequence of each probe molecule of the plurality of probe molecules is unique.

16. The method of claim 1 further comprising ligating capture tag sequences of the one or more cleaved probes to capture probes of the plurality of capture probes.

17. The method of claim 1 further comprising, prior to the cleaving, extending probe molecules that hybridize to nucleic acids of the sample.

18. The method of claim 1 wherein the cleaving comprises cleavage by a Type IIS restriction endonuclease.

19. The method of claim 1 wherein the cleaving comprises cleavage by a flap endonuclease.

11. The method of 10 wherein a derivative nucleic acid is a substrate or template for the enzyme mediated reaction.