Single molecule nucleic acid detection by mismatch cleavage
AssigneeStratos Genomics, Inc.
InventorsMark Stamatios Kokoris, John Tabone, Melud Nabavi, Cara Machacek
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Abstract
Methods and materials are provided for detecting nucleic acid sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions in single molecule target members present in a test population of nucleic acid fragments. Heteroduplexes are formed between members of the test nucleic acid population and their corresponding complements provided in a pool of mismatch cleavage probes. Mismatched base pairs in the heteroduplexes are specifically cleaved and cleaved probe fragments are electronically detected to signal the present of the target members in the test population.
Claims34
A method for determining at least one mutation or a polymorphism in a single molecule of a target sequence of a polynucleotide relative to a reference sequence of the polynucleotide comprising:
- (a) providing a test sample comprising a plurality of single-stranded polynucleotides;
- (b) providing a mismatch cleavage probe comprising:
- i. an oligonucleotide, wherein the oligonucleotide comprises a reference sequence, wherein the reference sequence comprises a sequence of the reverse complement of the single-stranded target nucleic acid and contains one or more nucleotide differences relative to the target nucleic acid, wherein the oligonucleotide is capable of hybridizing to the target nucleic acid to form a heteroduplex, wherein the heteroduplex comprises one or more base pair mismatches;
- ii. a first target identifier linked to the oligonucleotide 5′ to the position of the one or more nucleotide differences;
- and
- iii. a second target identifier linked to the oligonucleotide 3′ to the position of the one more nucleotide differences;
- wherein the first and second target identifiers are capable of generating distinct and reproducibly detectable signals
- (c) mixing the test sample with the mismatch cleavage probe under annealing conditions to form heteroduplexes between the mismatch cleavage probe and the target sequence;
- (d) contacting the heteroduplexes with a cleavage factor, wherein the cleavage factor is capable of cleaving mismatched bases in the heteroduplexes, wherein cleavage of the heteroduplex dissociates the first and second target identifiers of the mismatch cleavage probe;
- (e) optionally providing conditions to denature the heteroduplexes;
- and
- (f) determining the presence of the cleaved target sequence by electronically detecting the dissociation of the first and second target identifiers.
A mismatch cleavage probe for detecting a single molecule single-stranded target nucleic acid in a sample comprising:
- (a) an oligonucleotide, wherein the oligonucleotide comprises a reference sequence, wherein the reference sequence comprises a sequence of the reverse complement of the single-stranded target nucleic acid and contains one or more nucleotide differences relative to the target nucleic acid, wherein the oligonucleotide is capable of hybridizing to the target nucleic acid to form a heteroduplex, wherein the heteroduplex comprises one or more base pair mismatches;
- (b) a first target identifier linked to the oligonucleotide 5′ to the position of the one or more nucleotide differences, and
- (c) a second target identifier linked to the oligonucleotide 3′ to the position of the one more nucleotide differences;
- wherein the first and second target identifiers are capable of generating distinct and reproducibly detectable signals.
A circular mismatch cleavage probe for detecting a single molecule target nucleic acid in a sample comprising:
- (a) an oligonucleotide, wherein the oligonucleotide comprises a reference sequence, wherein the reference sequence comprises a sequence of the reverse complement of the single-stranded target nucleic acid and contains one or more nucleotide differences relative to the target nucleic acid, wherein the oligonucleotide is capable of hybridizing to the target nucleic acid to form a heteroduplex, wherein the heteroduplex comprises one or more base pair mismatches;
- (b) a target identifier linked to the 5′ end of the oligonucleotide, wherein the target identifier comprises a translocation control element and wherein the target identifier is capable of generating a distinct and reproducibly detectable signal upon passage through a nanopore;
- and
- (c) a leader sequence associated with a hydrophobic capture element, wherein the hydrophobic capture element is linked to the target identifier and the leader sequence is linked to the 3′ end of the oligonucleotide;
- wherein the circular mismatched cleavage probe is not capable of passage through a nanopore, wherein cleavage of the oligonucleotide linearizes the mismatch cleavage probe, and wherein the linear mismatch cleavage probe is capable of passage through a nanopore.
A method for amplifying a signal indicating at least one mutation or a polymorphism in a target sequence of a polynucleotide relative to a reference sequence of the polynucleotide comprising:
- (a) (Original) a mismatch cleavage stage, wherein the mismatch cleavage stage comprises contacting the target sequence with a mismatch amplifier probe and a mismatch endonuclease to produce a cleaved amplifier probe;
- (b) iterative rounds of a signal amplification stage, wherein a single round of the signal amplification stage comprises contacting the amplifier probe with a pool of amplification code probes and a nickase enzyme to produce a cleaved amplification code probe capable of producing a distinct and reproducible signal upon passage through a nanopore.
A mismatch amplifier probe for amplifying a signal indicating at least one mutation or a polymorphism in a target sequence of a polynucleotide relative to a reference sequence of the polynucleotide comprising:
- (a) an oligonucleotide, wherein the oligonucleotide comprises a reference sequence, wherein the reference sequence comprises a sequence of the reverse complement of the single-stranded target nucleic acid and contains one or more nucleotide differences relative to the target nucleic acid, wherein the oligonucleotide is capable of hybridizing to the target nucleic acid to form a heteroduplex, wherein the heteroduplex comprises one or more base pair mismatches;
- (b) a first hybridization oligonucleotide;
- (c) a first nickase recognition oligonucleotide;
- and
- (d) a biotin moiety.
An amplification code probe for amplifying a signal indicating at least one mutation or a polymorphism in a target sequence of a polynucleotide relative to a reference sequence of the polynucleotide comprising:
- (a) second hybridization oligonucleotide, wherein the sequence of the second hybridization oligonucleotide comprises the reverse complement of the sequence of the first hybridization oligonucleotide;
- (b) a second nickase recognition oligonucleotide, wherein the sequence of the second nickase recognition oligonucleotide comprises the reverse complement of the sequence of the first nickase recognition oligonucleotide, and wherein the second nickase recognition oligonucleotide is capable of being cleaved by a nickase endonuclease;
- (c) a target identifier;
- (d) a hydrophobic capture element;
- (e) a leader sequence;
- and
- (f) a streptavidin moiety.
A circular amplification code probe for amplifying a signal indicating at least one mutation or a polymorphism in a target sequence of a polynucleotide relative to a reference sequence of the polynucleotide comprising:
- (a) second hybridization oligonucleotide, wherein the sequence of the second hybridization oligonucleotide comprises the reverse complement of the sequence of the first hybridization oligonucleotide;
- (b) a second nickase recognition oligonucleotide linked to the 3′ end of the second hybridization oligonucleotide, wherein the sequence of the second nickase recognition oligonucleotide comprises the reverse complement of the sequence of the first nickase recognition oligonucleotide, and wherein the second nickase recognition oligonucleotide is capable of being cleaved by a nickase endonuclease;
- (c) a target identifier linked to the 5′ end of the second hybridization oligonucleotide;
- (d) a hydrophobic capture element linked to the 5′ end of the target identifier;
- and
- (e) a leader sequence linked to the 5′ end of the hydrophobic capture element and the 3′ end of the second nickase recognition oligonucleotide.
Dependencies are inferred from the claim text.