Methods for analyzing a target nucleic acid using immobilized heterogeneous mixtures of oligonucleotide probes

• US 6,309,824 B1
• Filed: 01/16/1997
• Issued: 10/30/2001
• Est. Priority Date: 01/16/1997
• Status: Expired due to Fees

• Current Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Original Assignee: Hyseq, Inc. (Arca Biopharma, Inc.)
• Inventors: Drmanac, Radoje T.
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US08/784,747
• Time in Patent Office: 1,748 Days
• Field of Search: 435/6, 435/91.5, 435/91.52, 436/501, 436/94, 536/24.31, 536/24.32, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• 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

Abstract

The present invention provides a method for detecting a target nucleic acid species including the steps of providing an array of probes affixed to a substrate and a plurality of labeled probes wherein each labeled probe is selected to have a first nucleic acid sequence which is complementary to a first portion of a target nucleic acid and wherein the nucleic acid sequence of at least one probe affixed to the substrate is complementary to a second portion of the nucleic acid sequence of the target, the second portion being adjacent to the first portion; applying a target nucleic acid to the array under suitable conditions for hybridization of probe sequences to complementary sequences; introducing a labeled probe to the array; hybridizing a probe affixed to the substrate to the target nucleic acid; hybridizing the labeled probe to the target nucleic acid; affixing the labeled probe to an adjacently hybridized probe in the array; and detecting the labeled probe affixed to the probe in the array.

Claims

1. A method for analyzing a target nucleic acid, comprising the steps of:

providing an array comprising a substrate and a plurality of heterogenous mixtures of different oligonucleotide probes, wherein each of said mixtures is immobilized on the substrate at a distinct position with the array;

providing a plurality of labeled oligonucleotide probes;

contacting the target nucleic acid with the immobilized probes and the labeled probes under conditions that allow the immobilized probes and the labeled probes which form perfectly hybridized matches with the target nucleic acid to be distinguished from the immobilized probes and the labeled probes that hybridize with a one base mismatch to the target nucleic acid;

covalently joining the immobilized probes and labeled probes that hybridize adjacent to each other with a target nucleic acid molecule

identifying a plurality of covalently joined immobilized probes and labeled probes; and

analyzing the nucleic acid from the identified probes.

2. The method of claim 1, wherein each of said mixtures is complementary to a plurality of overlapping sites in the target nucleic acid.

3. The method of claim 2, further comprising the steps of:

denaturing the covalently joined immobilized probes and labeled probes that hybridize to said target nucleic acid; and

repeating the contacting, covalently joining, and denaturing steps.

4. The method of claim 1, wherein each of said mixtures is complementary to a plurality of adjacent sites in the target nucleic acid.

5. The method of claim 4, further comprising the steps of:

denaturing the covalently joined immobilized probes and labeled probes that hybridize to said target nucleic acid; and

repeating the contacting, covalently joining, and denaturing.

6. The method of claim 1, wherein each of said mixtures is complementary to a plurality of nonadjacent sites in the target nucleic acid.

7. The method of claim 6, further comprising the steps of:

denaturing the covalently joined immobilized probes and labeled probes that hybridize to said target acid; and

repeating the contacting, covalently joining, and denaturing.

8. The method of claim 1, wherein each labeled probe is labeled with a plurality of labels.

9. The method of claim 8, wherein the labels are selected from the group consisting of a chromogenic label, an enzymatic label, and a radioactive label.

10. The method of claim 1, further comprising the step of checking a predicted sequence for the target nucleic acid versus a sequence obtained from the covalently joined probes.

17. The method of claim 1, wherein the immobilized probe and the labeled probe are covalently joined so that they will act as a single longer probe for purposes of branch point analysis.

11. A method of analyzing a target nucleic acid, comprising the steps of:

providing an array comprising a substrate and a plurality of heterogenous mixtures of different oligonucleotide probes, wherein each of said mixtures is immobilized on the substrate at a distinct position within the array;

providing a plurality of free oligonucleotide probes, wherein the free probes have a common tail;

contacting the target nucleic acid with the immobilized probes and free probes under conditions that allow the immobilized probes and the free probes which form perfectly hybridized matches with the nucleic acid to be distinguished from the probes that hybridize with a one base mismatch to the target nucleic acid;

covalently joining the immobilized probes and the free probes that hybridize adjacent to each other with a target nucleic acid molecule;

covalently joining the free probes with a probe agent that is multiply labeled, wherein the probe a;

gent specifically binds to the common tail of the free probe;

identifying a plurality of covalently joined immobilized probes and free probes that are covalently joined with the labeled probe agent; and

analyzing the nucleic acid from the identified probes.

12. The method of claim 11, wherein the target nucleic acid has a known sequence.

13. The method of claim 11, wherein a mixture of the probes is immobilized at a single distinct position within the array, and the target nucleic acid has a known sequence.

14. The method of claim 11, wherein the target nucleic acid has a known sequence, and further comprising the step of checking a predicted sequence for the target nucleic acid versus a sequence obtained from the covalently joined probes.

15. The method of claim 11, wherein the immobilized probe and the free probe are covalently joined so that they will act as a single longer probe for purposes of branch point analysis.

16. The method of claim 15, further comprising the step of checking a predicted sequence for the target nucleic acid versus a sequence obtained from the covalently joined probes.

18. A method for analyzing a target nucleic acid, comprising the steps of:

providing a substrate and a heterogenous mixture of different oligonucleotide probes, wherein the mixture is immobilized on the substrate at a distinct position within the substrate;

providing a plurality of labeled oligonucleotide probes;

contacting the target nucleic acid with the immobilized probes and the labeled probes under conditions that allow the immobilized probes and the labeled probes that form perfectly hybridized matches with the target nucleic acid to be distinguished from the immobilized probes and the labeled probes that hybridize with a one base mismatch to the target nucleic acid;

covalently joining the immobilized probes and labeled probes that hybridize adjacent to each other with a target nucleic acid molecule;

identifying a plurality of covalently joined immobilized probes and labeled probes; and

analyzing the nucleic acid from the identified probes.