Method and sequences for determinate nucleic acid hybridization
First Claim
1. A method of employing oligonucleotide probes to obtain information on a position of interest on a target sequence segment of a target nucleic acid analyte, the method comprising:
- contacting the target nucleic acid analyte, under hybridizing conditions, with at least two oligonucleotide probes having a variable position and are complementary with the target nucleic acid analyte, wherein on at least one of the at least two oligonucleotide probes, the variable position is occupied by a degenerately base pairing nucleotide analog selected from the group consisting of dP and 8-oxo-dG and on at least one other of the at least two oligonucleotide probes, the variable position is occupied by a degenerately base pairing nucleotide analog selected from the group consisting of dP and 8-oxo-dG or a non-degenerately base pairing nucleotide;
wherein hybridization of one or all of the at least two oligonucleotide probes to the target sequence segment occurs only if the degenerately base pairing nucleotide analog or the non-degenerately base pairing nucleotide at the variable position base pairs with a nucleotide at the position of interest on the target sequence segment; and
further wherein none of the at least two oligonucleotide probes hybridizes to the target nucleic acid analyte if there is a mismatch between the degenerately base pairing nucleotide analog or the non-degenerately base pairing nucleotide at the variable position and the nucleotide at the position of interest on the target sequence segment.
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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.
30 Citations
118 Claims
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1. A method of employing oligonucleotide probes to obtain information on a position of interest on a target sequence segment of a target nucleic acid analyte, the method comprising:
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contacting the target nucleic acid analyte, under hybridizing conditions, with at least two oligonucleotide probes having a variable position and are complementary with the target nucleic acid analyte, wherein on at least one of the at least two oligonucleotide probes, the variable position is occupied by a degenerately base pairing nucleotide analog selected from the group consisting of dP and 8-oxo-dG and on at least one other of the at least two oligonucleotide probes, the variable position is occupied by a degenerately base pairing nucleotide analog selected from the group consisting of dP and 8-oxo-dG or a non-degenerately base pairing nucleotide;
wherein hybridization of one or all of the at least two oligonucleotide probes to the target sequence segment occurs only if the degenerately base pairing nucleotide analog or the non-degenerately base pairing nucleotide at the variable position base pairs with a nucleotide at the position of interest on the target sequence segment; and
further wherein none of the at least two oligonucleotide probes hybridizes to the target nucleic acid analyte if there is a mismatch between the degenerately base pairing nucleotide analog or the non-degenerately base pairing nucleotide at the variable position and the nucleotide at the position of interest on the target sequence segment. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 31, 33, 34, 35, 36, 37, 117, 118)
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2-5. -5. (canceled)
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25-26. -26. (canceled)
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27. The method of 6 wherein the target nucleic acid analyte is sequenced by analysis of hybridization data obtained from the target sequence segments attached to a substrate surface.
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28. The method of 14 wherein the array is comprised of individual beads or particles, each bead or particle having a surface to which are attached a plurality of identical sequence segments.
- 29. The method of 14 wherein the array comprises an integrated substrate with a surface comprised of discrete sites, each site having a surface, the surface having attached a plurality of target sequence segments having an identical sequence.
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32. (canceled)
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40-115. -115. (canceled)
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116. (canceled)
Specification