Molecular detection systems utilizing reiterative oligonucleotide synthesis

A method of detecting multiple reiterated oligonucleotides from a target DNA or RNA polynucleotide, said method comprising:

• (a) hybridizing an initiator to a single-stranded target polynucleotide;
• (b) incubating said target polynucleotide and initiator with a target site probe, an RNA-polymerase, and a terminator, wherein said target site probe hybridizes with said target polynucleotide;
• (c) synthesizing an oligonucleotide transcript that is complementary to said target site from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript, thereby synthesizing multiple reiterative oligonucleotide transcripts;
• and
• (d) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.

A method for detecting methylated cytosine residues at CpG sites in a target polynucleotide, comprising:

• (a) deaminating a single-stranded target DNA sequence under conditions which convert unmethylated cytosine residues to uracil residues while not converting methylated cytosine residues to uracil;
• (b) hybridizing an initiator with a single stranded target polynucleotide;
• (c) incubating said deaminated target polynucleotide and said initiator with a terminator, and an RNA-polymerase, wherein at least one of said initiator, or terminator is modified to enable detection of the CG sites;
• (d) synthesizing an oligonucleotide transcript that is complementary to said CG sites from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript thereby synthesizing multiple reiterative oligonucleotide transcripts;
• and
• (e) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.

A method for detecting methylated cytosine residues at a CpG site in a target gene, said method comprising:

• (a) deaminating a single-stranded target DNA polynucleotide under conditions which convert unmethylated cytosine residues to uracil residues while not converting methylated cytosine residues to uracil;
• (b) hybridizing a target site probe with said single stranded target polynucleotide,
• (c) incubating said target polynucleotide and target site probe with, an initiator, a terminator, and an RNA-polymerase, wherein at least one of said initiator, or said terminator are complementary to the CpG site;
• (d) synthesizing an oligonucleotide transcript that is complementary to said target site from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides, thereby synthesizing multiple reiterative oligonucleotide transcripts;
• and
• (e) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.

A method for detecting the presence or absence of mutations in a target DNA sequence, the method comprising

• (a) hybridizing a target site probe to a single-stranded DNA polynucleotide, wherein said DNA polynucleotide may contain a mutation relative to a normal or wild type gene;
• (b) incubating said target polynucleotide and target-site probe with an RNA-polymerase, an initiator, and a terminator;
• (c) synthesizing an oligonucleotide transcript from said target polynucleotide that is complementary to a target mutation site, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple abortive reiterative oligonucleotides;
• and
• (d) determining the presence or absence of a mutation by detecting or quantifying said reiteratively synthesized oligonucleotides transcribed from said target DNA polynucleotide.

A method for detecting mutations in a target DNA polynucleotide, said method comprising:

• (a) immobilizing a capture probe designed to hybridize with said target DNA polynucleotide;
• (b) hybridizing said capture probe to said target DNA polynucleotide, wherein said DNA polynucleotide may contain a mutation relative to a normal or wild type gene;
• (c) incubating said target polynucleotide with an RNA-polymerase, an initiator, and a terminator;
• (d) synthesizing an oligonucleotide transcript that is complementary to a target site from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript, thereby synthesizing multiple abortive reiterative oligonucleotide transcripts;
• and
• (e) determining the presence or absence of a mutation by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts from said target DNA polynucleotide.

A method for detecting DNA or RNA in a test sample, said method comprising:

• (a) hybridizing a single stranded target polynucleotide with an abortive promoter cassette comprising a sequence that hybridizes to the single stranded target polynucleotide, and a region that can be detected by transcription by a polymerase;
• (b) incubating said target polynucleotide with an RNA-polymerase, an initiator, and a terminator;
• (c) synthesizing an oligonucleotide transcript that is complementary to the initiation start site of the APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides, thereby synthesizing multiple reiterative oligonucleotide transcripts;
• and
• (d) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.

A method for detecting the presence of pathogens in a test sample, said method comprising the steps of:

• (a) hybridizing a single stranded target pathogen polynucleotide in said test sample with an abortive promoter cassette comprising a region that can be detected by transcription by a polymerase;
• (b) incubating said target polynucleotide and initiator with an RNA-polymerase, and a terminator;
• (c) synthesizing an oligonucleotide transcript that is complementary to initiation start site of the APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple abortive reiterative oligonucleotide transcripts;
• and
• (d) determining the presence of a pathogen by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts synthesized from said test sample.

A method for detecting pathogens in a test sample, said method comprising:

• (a) immobilizing a capture probe designed to hybridize with a target DNA polynucleotide in said test sample;
• (b) hybridizing said capture probe with a test sample that potentially contains said target DNA polynucleotide;
• (c) hybridizing a single stranded target DNA polynucleotide in said test sample with an abortive promoter cassette comprising a region that hybridizes to the single stranded target pathogen polynucleotide, and a region that can be detected by transcription by a polymerase;
• (d) incubating said target polynucleotide with an RNA-polymerase, initiator, and a terminator;
• (e) synthesizing an oligonucleotide transcript that is complementary to said initiation transcription start site of APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple reiterative oligonucleotide transcripts;
• and
• (f) determining the presence or absence of a pathogen by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.

A method for detecting mRNA expression in a test sample, the method comprising:

• (a) hybridizing a target mRNA sequence with an abortive promoter cassette comprising a region that can be detected by transcription by a polymerase;
• (b) incubating said target mRNA sequence with an RNA-polymerase, an initiator, and a terminator;
• (c) synthesizing an oligonucleotide transcript that is complementary to transcription initiation start site, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript, thereby synthesizing multiple reiterative oligonucleotides;
• and
• (d) determining the presence or absence of the mRNA by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts synthesized from said test sample.

A method for detecting an oligonucleotide synthesized from a target DNA sequence, the method comprising:

• (a) hybridizing a DNA primer with a single-stranded target DNA sequence;
• (b) extending said DNA primer with a DNA polymerase and nucleotides, such that said DNA polymerase reiteratively synthesizes a nucleotide sequence;
• and
• (c) detecting oligonucleotide comprised of repeat sequences synthesized by said DNA polymerase.

A method for detecting multiple reiterated oligonucleotides from a target DNA or RNA polynucleotide, said method comprising:

• (a) incubating a single-stranded target polynucleotide in a mixture comprising an initiator, and an RNA-polymerase;
• (b) synthesizing multiple oligonucleotide transcripts from said target polynucleotide, wherein said initiator is extended until terminated due to nucleotide deprivation, thereby synthesizing multiple abortive reiterative oligonucleotide transcripts;
• and
• (c) detecting or quantifying said reiteratively synthesized oligonucleotides

A method of detecting multiple reiterated oligonucleotides from a target DNA or RNA polynucleotide, said method comprising:

• (a) incubating a single-stranded target polynucleotide in a mixture comprising an initiator, an RNA-polymerase, and a target site probe, wherein said target site probe and said target polynucleotide hybridize to form a bubble complex comprising a first double-stranded region upstream of a target site, a single-stranded region comprising said target site, and a second double-stranded region downstream of said target site;
• (b) synthesizing multiple oligonucleotide transcripts from said target polynucleotide, wherein said initiator is extended until terminated due to nucleotide deprivation, thereby synthesizing multiple abortive reiterative oligonucleotides;
• and
• (c) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts.

A method for detecting methylated cytosine residues at a CG site near a target gene, the method comprising:

• (a) deaminating a single-stranded target DNA sequence under conditions which convert unmethylated cytosine residues to uracil residues while not converting methylated cytosine residues to uracil;
• (b) incubating a single-stranded target polynucleotide in a mixture comprising an initiator, a terminator, an RNA-polymerase, and a target site probe;
• (c) synthesizing multiple oligonucleotide transcripts from said target polynucleotide, wherein said initiator is extended until terminated due to nucleotide deprivation, thereby synthesizing multiple abortive reiterative oligonucleotide transcripts;
• and
• (d) detecting or quantifying said reiteratively synthesized oligonucleotides

A method for detecting a target protein in a test sample, the method comprising:

• (a) covalently attaching the target protein to an abortive promoter cassette (APC) by a reactive APC linker, wherein said APC comprises a region that can be detected by transcription by a polymerase;
• (b) incubating said target protein with an RNA-polymerase, an initiator, and a terminator;
• (c) synthesizing an oligonucleotide transcript that is complementary to transcription initiation start site of APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript, thereby synthesizing multiple reiterative oligonucleotide transcripts;
• and
• (d) determining the presence or absence of the target protein by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts synthesized from said test sample.

A method for detecting cancer, comprising:

• (a) obtaining a sample from a patient in need of detection of a cancer;
• (b) deaminating the DNA under conditions which convert unmethylated cytosine residues to uracil residues while leaving the methylated cytosine residues unaltered;
• (c) hybridizing an initiator to a target polynucleotide wherein said initiator is a mononucleoside, mononucleotide, binucleotide, oligonucleotide or an analog thereof;
• (d) incubating said deaminated target polynucleotide and said initiator with a terminator, and an RNA-polymerase, wherein at least one of said initiator, terminator is modified to enable detection of the CG sites;
• (e) synthesizing an oligonucleotide transcript that is complementary to said CG sites from said target polynucleotide, wherein said initiator is extended until said terminator is incorporated into said oligonucleotide transcript thereby synthesizing multiple reiterative oligonucleotide transcripts;
• (f) detecting or quantifying said reiteratively synthesized oligonucleotide transcripts;
• and comparing the results with those obtained similarly from a control sample.

A method for detecting pathogens, said method comprising the steps of:

• (a) obtaining a sample in need of detection of a pathogen
• (b) hybridizing a single stranded target pathogen polynucleotide in said sample with an abortive promoter cassette comprising a nucleotide sequence that hybridizes to single stranded target pathogen polynucleotide, and a region that can be detected by transcription by a polymerase;
• (c) incubating said target polynucleotide and initiator with an RNA-polymerase, and a terminator;
• (d) synthesizing an oligonucleotide transcript that is complementary to initiation start site of the APC, wherein said initiator is extended until said terminator is incorporated into said oligonucleotides thereby synthesizing multiple abortive reiterative oligonucleotide transcripts;
• and
• (e) determining the presence of a pathogen by detecting or quantifying said reiteratively synthesized oligonucleotide transcripts synthesized from said sample.