Solid support assay systems and methods utilizing non-standard bases

A method of assaying a target oligonucleotide in a sample comprising steps of:

• (a) contacting a capture oligonucleotide coupled to a support with the sample under suitable hybridization conditions to hybridize a target oligonucleotide, the target oligonucleotide comprising a tagging sequence comprising at least one non-standard base and an analyte-specific sequence or a complement thereof, the capture oligonucleotide comprising a molecular recognition sequence comprising a sequence that is the same as or complementary to the analyte-specific sequence;
• (b) enzymatically extending the capture oligonucleotide using the target oligonucleotide as a template and incorporating a complementary non-standard base opposite the non-standard base of the tagging sequence;
• and
• (c) incorporating a reporter or coupling moiety onto an extended portion of the capture oligonucleotide, and
• (d) detecting the presence of the target oligonucleotide in the sample by detecting incorporation of the reporter or coupling moiety.

A method of assaying a target oligonucleotide comprising steps of:

• (a) contacting an analyte comprising the analyte-specific sequence with a first primer and a second primer, the first primer comprising a tagging sequence and a sequence complementary to a first sequence of the analyte, the second primer comprising a sequence complementary to a second sequence of the analyte and a non-standard base;
• (b) enzymatically extending the first and second primers to form a target oligonucleotide and a second oligonucleotide, wherein one of the target oligonucleotide and the second oligonucleotide comprises the analyte-specific sequence and the other comprises a sequence complementary to the analyte-specific sequence, wherein the extension of the first primer is substantially halted at the position opposite the non-standard base of the second primer;
• (c) incorporating a complementary non-standard base into the extended first primer opposite the non-standard base of the second primer;
• (d) contacting a capture oligonucleotide coupled to a support with the target oligonucleotide under hybridizing conditions to hybridize a target oligonucleotide, the target oligonucleotide comprising the tagging sequence and the analyte-specific sequence or a complement of the analyte-specific sequence, the capture oligonucleotide comprising a molecular recognition sequence that is the same as or complementary to at least a portion of the analyte-specific sequence;
• and
• (e) detecting hybridization of target oligonucleotide to the capture oligonucleotide.

A method of simultaneously detecting at least two alleles in a sample comprising genomic DNA comprising the steps of:

• (a) contacting the sample with at least two primer pairs comprising a first primer and a second primer under conditions such that each first and second primer of the primer pairs hybridizes to the genomic DNA;
• (b) amplifying the DNA sequences flanked by the first and second primer of each primer pair;
• (c) hybridizing the amplified DNA sequences of step (b) with at least two tagged, allele-specific primers, each tagged, allele specific primer comprising, in 5′ to 3′ order, a 5′ tagging seqeunce comprising at least one non-standard nucleotide, a linker, and a 3′ sequence capable of hybridizing with an amplified sequence of step (b);
• (d) enzymatically extending the allele-specific primer of step (c) in the presence of a labeled triphosphate base to form labeled extension products;
• (e) contacting the extension products of step (d) with at least two capture oligonucleotides coupled to a support under suitable hybridization conditions to hybridize an extension product, the extension product comprising a tagging sequence comprising at least one non-standard base and an allele-specific sequence, the capture oligonucleotide comprising a molecular recognition sequence comprising a sequence complementary to the tagging sequence, the molecular recognition sequence comprising a non-standard base complementary to the non-standard base of the;
• and
• (f) detecting the hybridization of at least to extension product to at least two capture oligonucleotides.

A method of assaying a target oligonucleotide comprising steps of:

• (a) contacting an analyte comprising the analyte-specific sequence with a first primer and a second primer, the second primer comprising, in 5′ to 3′ order, a non-complementary sequence, a non-standard base, and an analyte-specific sequence;
• (b) enzymatically extending the primers to form a target oligonucleotide and a second oligonucleotide, wherein one of the target oligonucleotide and the second oligonucleotide comprises the analyte-specific sequence and the other comprises a sequence complementary to the analyte-specific sequence;
• (c) hybridizing to the extension product of the second primer of step (b) a tagged, allele-specific primer comprising, in 5′ to 3′ order, a 5′ tagging sequence comprising a non-standard base, a linker, and a 3′ sequence complementary to the extension product of the second primer;
• (d) enzymatically extending the allele-specific primer of step (c);
• (e) hybridizing a reporter oligonucleotide complementary to the 5′ non-complementary sequence of the second primer of step (a) to the extension product of step (d), the reporter oligonucleotide comprising a 5′ phosphate and a reporter moiety, to form a nick structure suitable for ligation by a ligase;
• (f) contacting the nick structure of step (e) with a ligase to ligate the reporter oligonucleotide to the allele-specific extension product;
• (g) contacting a capture oligonucleotide coupled to a support with the ligation product of step (f);
• and
• (h) detecting hybridization of ligated oligonucleotide to the capture oligonucleotide

A kit for assaying an analyte, the kit comprising:

• a support,
• capture oligonucleotides coupled to the support, the capture oligonucleotides comprising a molecular recognition sequence having at least one non-standard base;
• first primers comprising a tagging sequence and a sequence complementary to a first sequence of the analyte;
• and
• second primers comprising a sequence complementary to a second sequence of the analyte.