Nucleic acid sequence determination by multiple mixed oligonucleotide probes

• US 5,002,867 A
• Filed: 10/24/1988
• Issued: 03/26/1991
• Est. Priority Date: 04/25/1988
• Status: Expired due to Fees

• Current Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Original Assignee: Stephen C. Macevicz
• Inventors: Macevicz, Stephen C.
• Primary Examiner(s): Yarbrough, Amelia Burgess
• Assistant Examiner(s): Fleisher, Mindy B.
• Application Number:
  US07/261,702
• Time in Patent Office: 883 Days
• Field of Search: 435/6, 435/810, 536/27, 935/77, 935/78, 436/501, 436/808
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• 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 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 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

A method is provided for sequencing nucleic acids without the need to separate similarly sized DNAs or RNAs by gel electrophoresis. The method relies on the separate hybridization of multiple mixed oligonucleotide probes to a target sequence. The mixed oligonucleotide probes comprise sequences of fixed and non-fixed bases corresponding to every possible permutation of fixed and non-fixed bases less than or equal to the length of the probes. For each probe, the hybridizations provide the number of times the probe'"'"'s particular sequence of fixed bases appears in the target sequence. The target sequence is then mathematically reconstructed from this data and a knowledge of the probe sequences.

Claims

1. A method for determining the nucleotide sequence of a nucleic acid, the method comprising the steps of:

providing a set of probes, each probe within the set having a predetermined length and each probe within the set having a predetermined sequence of fixed and non-fixed positions, the fixed positions comprising one or more predetermined kinds of nucleotides;

hybridizing the probes of the set to the nucleic acid;

determining the number of copies of each probe in the set that form perfectly matched duplexes with the nucleic acid; and

reconstructing the nucleotide sequence of the nucleic acid from the predetermined sequences of the probes that form perfectly matched duplexes with the nucleic acid.

2. The method of claim 1 wherein said set contains at least one probe comprising a sequence of fixed and nonfixed positions equivalent to that of each permutation of a plurality of fixed and non-fixed positions equal to or less than the length of the probe.

3. The method of claim 2 wherein said step of hybridizing includes:

anchoring a known quantity of said nucleic acid to each of a plurality of solid phase supports; and

washing each of the solid phase supports so that said probes forming perfectly matched duplexes with said nucleic acid are detectable.

4. The method of claim 3 wherein said step of hybridizing includes separately hybridizing each probe of said set to said nucleic acid on a different solid phase support of said plurality of solid phase supports.

5. The method of claim 4 wherein said predetermined length of said probes are in the range of from seven to eleven nucleotides, inclusive.

6. The method of claim 5 wherein said non-fixed positions of said probes are occupied by at least one degeneracy-reducing analog.

7. The method of claim 6 wherein said degeneracy-reducing analog is deoxyinosine whenever said fixed positions are occupied by nucleotides containing either cytosine or adenosine.

8. The method of claim 6 wherein said degeneracy-reducing analog is 2-aminopurine whenever said fixed positions are occupied by nucleotides containing either cytosine or guanosine.

9. The method of claim 6 wherein said degeneracy-reducing analog is N⁴ -methoxydeoxycytidine or 5-fluorodeoxyuridine whenever said fixed positions are occupied by nucleotides containing either adenosine or guanosine.

10. The method of claim 6 wherein said degeneracy-reducing analog minimizes the differences in binding energy between basepairs.

11. A kit for determining the nucleotide sequence of a nucleic acid, the kit comprising a set of oligonucleotides, each oligonucleotide within the set having a predetermined length and each oligonucleotide within the set having a predetermined sequence of fixed and non-fixed positions, the fixed positions comprising one or more predetermined kinds of nucleotides, and the set containing at least one probe comprising a sequence of fixed and non-fixed positions equivalent to that of each permutation of a plurality of fixed and non-fixed positions less than or equal to the length of the probe.

12. The kit of claim 11 further including a labelling means for detecting oligonucleotides capable of forming perfectly matched duplexes with said nucleic acid.

13. The kit of claim 12 further including a plurality of solid phase supports for anchoring said nucleic acid.

14. The kit of claim 13 further including a prehybridization solution for treating said solid phase supports to minimize nonspecific binding of said oligonucleotides to said solid phase supports.

15. The kit of claim 14 further including a set of maximally non-complementary oligonucleotides for each oligonucleotide of said set, the maximally non-complementary oligonucleotides being used with said prehybridization solution to block nonspecific binding sites of said oligonucleotides.

16. The kit of claim 12 wherein said labelling means includes a radioactive isotope covalently attached to said oligonucleotides.

17. The kit of claim 12 wherein said labelling means includes a fluorescent dye or an enzyme capable of generating a colorimetric signal.

18. The kit of claim 17 wherein said labelling means includes biotin conjugated to said oligonucleotides and includes avidin or streptavidin conjugated to said fluorescent dye or said enzyme capable of generating a colorimetric signal.

19. A method for determining the nucleotide sequence of a nucleic acid, the method comprising the steps of:

providing a first set of probes, each probe within the first set having the same length, the length being from seven to ten nucleotides, and each probe within the first set having a predetermined sequence of fixed and non-fixed bases, the fixed bases being deoxyadenosine and the non-fixed bases comprising deoxycytosine, deoxyguanosine, thymidine, or a degeneracy-reducing analog thereof, such that for each permutation of fixed and non-fixed bases less than or equal to the length of the probe, the first set contains at least one probe having a sequence equivalent to such permutation;

providing a second set of probes, each probe within the second set having the same length, the length being from seven to ten nucleotides, and each probe within the second set having a predetermined sequence of fixed and non-fixed bases, the fixed bases being deoxycytosine and the non-fixed bases comprising deoxyadenosine, deoxyguanosine, thymidine, or a degeneracy-reducing analog thereof, such that for each permutation of fixed and non-fixed bases less than or equal to the length of the probe, the second set contains at least one probe having a sequence equivalent to such permutation;

providing a third set of probes, each probe within the third set having the same length, the length being from seven to ten nucleotides, and each probe within the third set having a predetermined sequence of fixed and non-fixed bases, the fixed bases being deoxyguanosine and the non-fixed bases comprising deoxyadenosine, deoxycytosine, thymidine, or a degeneracy-reducing analog thereof, such that for each permutation of fixed and non-fixed bases less than or equal to the length of the probe, the third set contains at least one probe having a sequence equivalent to such permutation;

providing a fourth set of probes, each probe within the fourth set having the same length, the length being from seven to ten nucleotides, and each probe within the fourth set having a predetermined sequence of fixed and non-fixed bases, the fixed bases being thymidine and the non-fixed bases comprising deoxyadenosine, deoxycytosine, deoxyguanosine, or a degeneracy-reducing analog thereof, such that for each permutation of fixed and non-fixed bases the length of the probe, the fourth set contains at least one probe having a sequence equivalent to such permutation;

anchoring a known quantity of the nucleic acid to each of a plurality of solid phase supports;

separately hybridizing each probe of the first, second, third, and fourth sets to the nucleic acid anchored on the solid phase supports;

washing each of the solid phase supports after hybridizing said probes so that said probes forming perfectly matched duplexes with said nucleic acid are detectable;

determining the number of copies of each probe in each set that form perfectly matched duplexes with the nucleic acid; and

reconstructing the nucleotide sequence of the nucleic acid from the predetermined sequences of the probes that form perfectly matched duplexes with the nucleic acid.

20. The method of claim 19 wherein said nucleic acid contains at least one known sequence region.

21. The method of claim 20 wherein said probe of said first set having said length from eight to nine nucleotides, said probe of said second set having said length from eight to nine nucleotides, said probe of said third set having said length from eight to none nucleotides, and said probe of said fourth set having said length from eight to nine nucleotides.

22. The method of claim 21 wherein the said degeneracy-reducing analog of said first set includes deoxyinosine, 5-fluorodeoxyuridine, and N⁴ -methoxycytosine, said degeneracy-reducing analog of said second set includes deoxyinosine and 2-aminopurine, and said degeneracy-reducing analog of said third set includes 2-aminopurine and N⁴ -methoxycytosine.

23. The method of claim 22 wherein said step of washing includes exposing each one of said plurality of solid phase supports to tetramethylammonium chloride at a concentration of between about 2 to 4 moles per liter.