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US 2025/0093341 A1Published application
Method for relative quantification of nucleic acid sequence, expression, or copy changes, using combined nuclease, ligation, and polymerase reactions
AssigneeCornell University
InventorsFrancis BARANY, Eugene SPIER, Alain MIR
Life of this patent
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Abstract
The present invention is directed to kits for identifying the presence of one or more target nucleotide sequences in a sample that involve a ligation and/or polymerase reaction. In some embodiments, the ligation products formed in the ligation process of the present invention are subsequently amplified using a polymerase chain reaction. The ligated product sequences or extension products thereof are detected, and the presence of one or more target nucleotide sequences in the sample is identified based on the detection.
Claims79
A method for identifying a presence of one or more target nucleotide sequences in a sample comprising:
- providing a sample potentially containing the one or more target nucleotide sequences;
- providing one or more oligonucleotide probe sets, each set comprising (a) a first oligonucleotide probe having a target-specific portion, and (b) a second oligonucleotide probe having a target specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize adjacent to one another on the target nucleotide sequence with a junction between the first and second oligonucleotide probes, and wherein, in a probe set, the target specific portion of the second oligonucleotide probe has an overlapping identical nucleotide at the junction with the first oligonucleotide probe;
- contacting the sample and the one or more oligonucleotide probe sets under conditions effective for first and second oligonucleotide probes of a probe set to hybridize at adjacent positions in a base specific manner to their corresponding target nucleotide sequences, if present in the sample, wherein upon hybridization the overlapping identical nucleotide of the second oligonucleotide probe forms a flap at the junction comprising the overlapping identical nucleotide;
- cleaving the overlapping identical nucleotide of the second oligonucleotide probe with an enzyme having 5′ nuclease activity, thereby liberating a phosphate at the second oligonucleotide probe's 5′end;
- ligating first and second oligonucleotide probes of the one or more oligonucleotide probe sets together at the junction to form ligated product sequences;
- detecting the ligated product sequences in the sample;
- and
- identifying the presence of one or more target nucleotide sequences in the sample based on said detecting.
5 more elements
The method of claim 23, wherein the solid support is in a form selected from the group consisting of beads, slides, discs, membranes, films, microtiter plates, and composites thereof.
A method for identifying a presence of one or more target nucleotide sequences in a sample, said method comprising:
- providing a sample potentially containing the target nucleotide sequence;
- providing one or more oligonucleotide probe sets, each set comprising (a) a first oligonucleotide probe having a target-specific portion, and (b) a second oligonucleotide probe having 5′ non-target specific flap portion and a target-specific portion containing one or more thiophosphate-modified nucleotide bases, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize on the target nucleotide sequence;
- contacting the sample and the one or more oligonucleotide probe sets under conditions effective for first and second oligonucleotide probes of a probe set to hybridize in a base specific manner to their corresponding target nucleotide sequences, if present in the sample;
- cleaving the 5′ non-target specific flap portion of the second oligonucleotide probe with an enzyme having 5′ nuclease activity, thereby liberating a 5′ phosphate at a first nucleotide base of the target-specific portion of the second oligonucleotide;
- ligating first and second oligonucleotide probes of the one or more oligonucleotide probe sets together to form ligated product sequences containing the target-specific portions with the one or more thiophosphate-modified nucleotide bases;
- detecting ligated product sequences in the sample;
- and
- identifying the presence of the one or more target nucleotide sequences in the sample based on said detecting.
5 more elements
A method for identifying a presence of one or more target nucleotide sequences in a sample comprising:
- providing a sample potentially containing the one or more target nucleotide sequences;
- providing one or more oligonucleotide probe sets, each set comprising (i) a first oligonucleotide probe comprising a 5′ primer-specific portion, a first portion of a zip-code portion, a first tag portion that is 3′ to the first zip-code portion, and a target-specific portion, and (ii) a second oligonucleotide probe comprising a 3′ primer-specific portion, a second portion of the zip-code portion, a second tag portion that is 5′ to the second zip-code portion, and a target-specific portion, wherein the first and second zip-code portions of an oligonucleotide probe set, when adjacently positioned, form a full-length zip-code, and wherein the first and second tag portions of an oligonucleotide probe set are complementary to each other;
- contacting the sample and the one or more oligonucleotide probe sets under conditions effective for first and second oligonucleotide probes of a probe set to hybridize in a base specific manner to their corresponding target nucleotide sequences, if present in the sample;
- ligating first and second oligonucleotide probes of the one or more probe sets together to form ligated product sequences;
- providing one or more oligonucleotide primer sets, each set comprising (a) a first oligonucleotide primer comprising the same nucleotide sequence as the 5′ primer-specific portion of the ligated product sequence and (b) a second oligonucleotide primer comprising a nucleotide sequence that is complementary to the 3′ primer-specific portion of the ligated product sequence;
- blending the ligated product sequences, the one or more oligonucleotide primer sets, and a DNA polymerase to form a polymerase chain reaction mixture;
- subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles thereby forming primary extension products;
- providing a collection of capture oligonucleotides complementary to a portion of the first zip-code portion and a portion of the second zip-code portion, wherein each capture oligonucleotide of the collection comprises a quencher molecule and a detectable label separated from each other;
- subjecting the primary extension products and the collection of capture oligonucleotides to conditions effective for (i) the first and second tag portions of a particular primary extension product to hybridize to each other to form hairpinned extension products with adjacently positioned first and second zip-code portions and (ii) the capture oligonucleotides of the collection to hybridize to complementary adjacently positioned first and second zip-code portions of the hairpinned extension products;
- cleaving the quencher molecule or the detectable label from the hybridized capture oligonucleotides;
- detecting the detectable label separated from the quencher molecule;
- and
- identifying the presence of the one or more target nucleotide sequences in the sample based on said detecting.
10 more elements
A method for identifying a presence of one or more target nucleotide sequences in a sample comprising:
- providing a sample potentially containing the one or more target nucleotide sequences;
- providing one or more oligonucleotide probe sets, each set comprising (i) a first oligonucleotide probe comprising a 5′ primer-specific portion, a first portion of a zip-code portion, a first tag portion that is 3′ to the first zip-code portion, and a target-specific portion, and (ii) a second oligonucleotide probe comprising a 3′ primer-specific portion, a second portion of the zip-code portion, a second tag portion that is 5′ to the second zip-code portion and a target-specific portion, wherein the first and second zip-code portions of an oligonucleotide probe set, when adjacently positioned, form a full-length zip-code, and wherein the first and second tag portions of an oligonucleotide probe set are complementary to each other;
- contacting the sample and the one or more oligonucleotide probe sets under conditions effective for first and second oligonucleotide probes of a probe set to hybridize in a base specific manner to their corresponding target nucleotide sequences, if present in the sample;
- ligating first and second oligonucleotide probes of the one or more probe sets together to form ligated product sequences;
- providing one or more oligonucleotide primer sets, each set comprising (i) a first oligonucleotide primer having (a) a nucleotide sequence that is the same as the second primer-specific portion of the first oligonucleotide probe, (b) a capture oligonucleotide portion that is complementary to adjacently positioned first and second zip-code portions of an oligonucleotide probe set, (c) a quencher molecule and a detectable label separated by said capture oligonucleotide portion, (ii) a second oligonucleotide primer comprising a nucleotide sequence that is complementary to the 3′ primer-specific portion of the ligated product sequence;
- blending the ligated product sequences, the one or more oligonucleotide primer sets, and a DNA polymerase to form a polymerase chain reaction mixture;
- subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles thereby forming primary extension products;
- subjecting the primary extension products to conditions effective for the first and second tag portions of a particular primary extension product to hybridize to each other to form hairpinned primary extension products with adjacently positioned first and second zip-code portions and (ii) the capture oligonucleotide portion of a particular hairpinned primary extension product to hybridize to complementary adjacently positioned first and second zip-code portions of the hairpinned extension product;
- cleaving the quencher molecule or the detectable label from capture oligonucleotide portion of the hairpinned primary extension products;
- detecting the detectable label separated from the quencher molecule;
- and
- identifying the presence of the one or more target nucleotide sequences in the sample based on said detecting.
9 more elements
A kit for identifying a presence of one or more target nucleotide sequences in a sample comprising:
- an enzyme having 5′ nuclease activity;
- a ligase;
- and
- one or more oligonucleotide probe sets, each set comprising (a) a first oligonucleotide probe having a target-specific portion, and (b) a second oligonucleotide probe having a target specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize adjacent to one another on the target nucleotide sequence with a junction between the first and second oligonucleotide probes, and wherein, in a probe set, the target specific portion of the second oligonucleotide probe has an overlapping identical nucleotide at the junction with the first oligonucleotide probe.
1 more element
A kit for identifying a presence of one or more target nucleotide sequences in a sample comprising:
- an enzyme having 5′ nuclease activity;
- a ligase;
- and
- one or more oligonucleotide probe sets, each set comprising (a) a first oligonucleotide probe having a target-specific portion, and (b) a second oligonucleotide probe having 5′ non-target specific flap portion and a target-specific portion containing one or more thiophosphate-modified nucleotide bases, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize on the target nucleotide sequence.
1 more element
Dependencies are inferred from the claim text.