Method and sequences for determinate nucleic acid hybridization

• US 20070042389A1
• Filed: 08/16/2005
• Published: 02/22/2007
• Est. Priority Date: 03/28/2001
• Status: Active Grant

• Current Assignee: William Hillis
• Original Assignee: William Hillis
• Inventors: Hillis, William
• Granted Patent: US 7,537,892 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/117 incorporating modified base

Abstract

Provided are methods for using nucleic acid sequences having two or more degenerately pairing nucleotides, each degenerate nucleotide having a partially overlapping set of complementarity, to reduce the number of hybridizing nucleotide sequences or probes used in biochemical and molecular biological operations having sequence specific hybridization. The method may be employed for various hybridization procedures with sequence specific hybridization, including sequencing methods measuring hybridization directly, and tagging by hybridization methods in which the sequence is determined by analyzing the pattern of tags that hybridize thereto, and hybridization dependent amplification methods. The method involves hybridizing to the nucleic acid sequence of interest a first hybridizing nucleotide sequence and a second hybridizing nucleotide sequence, each comprising a sequence complementary, or complementary except at a position of interest or variable position, to a nucleic acid sequence of interest, and analyzing the whether some, all or none of the probes or tags hybridize.

Claims

1. A method of determining the identity of an unknown nucleotide at a position of interest on a target nucleic acid analyte comprising:

contacting the nucleic acid analyte under hybridizing conditions with two oligonucleotide probes having at least one nucleotide in common and having a variable position that is independently selected from the group consisting of dPTP, 8-oxo-dGTP, A, T, C, and G if the target nucleic acid analyte is DNA, and dPTP, 8-oxo-dGTP, A, U, C, and G if the target nucleic acid analyte is RNA,

wherein the identity of the position of interest is determined by comparing the variable positions of the hybridized probes for complementarity and the variable positions of the nonhybridized probes for lack of complementarity.

2. The method of claim 1 wherein dPTP hybridizes to A and G and 8-oxo-dGTP hybridizes to A and C.

3. The method of claim 2 wherein the variable position of one of the oligonucleotide probes is occupied by dPTP and the variable position of the other oligonucleotide probe is occupied by 8-oxo-dGTP.

4. The method of claim 2 wherein nonhybridization indicated T or U at the position of interest.

5. The method of claim 2 wherein hybridization of both probes indicates A at the position of interest.

6. The method of claim 2 wherein hybridization of dPTP but not 8-oxo-dGTP indicates G at the position of interest.

7. The method of claim 2 wherein hybridization of 8-oxo-dGTP but not dPTP indicates C at the position of interest.

8. The method of claim 1 wherein the variable position of one of the oligonucleotide probes is occupied by T, U, or C and the variable position of the other oligonucleotide probe is occupied by T or G.

9. The method of claim 8 wherein hybridization of both probes indicates A at the position of interest.

10. The method of claim 8 wherein hybridization of neither probe indicates T at the position of interest.

11. The method of claim 8 wherein hybridization of the probe having T, U, or C at the variable position but no hybridization of the probe having T or G at the variable position indicates G at the position of interest.

12. The method of claim 8 wherein hybridization of the probe having T, U, or C at the variable position but no hybridization of the probe having T, U, or C at the variable position indicates C at the position of interest.