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US 10,472,671 B2Granted
Workflow for detection of ligands using nucleic acids
AssigneeLife Technologies Corporation
InventorsShiaw-Min Chen, Elana Swartzman, David Ruff, Mark Shannon, Julia Lu, Stephen Hendricks
Life of this patent
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Abstract
This application relates to methods for ligating oligonucleotides having complementarity to a target nucleic acid, and amplifying the ligated oligonucleotides, where ligation and amplification occur in the same reaction mixture.
Claims17
A method for performing a proximity ligation assay on a test sample comprising:
- a) contacting a target protein or analyte with at least a first probe and a second probe, each probe having binding specificity for the protein or analyte, and being adjoined to at least one type of DNA oligonucleotide to form a binding reaction mixture;
- b) contacting the binding reaction mixture with a ligation-PCR reaction mixture comprising:
- (i) a small-footprint DNA ligase (SFL) selected from the group consisting of a ligase of SEQ ID NO.:
- 77, a ligase of SEQ ID NO.:
- 78, a ligase of SEQ ID NO.:
- 79, a ligase of SEQ ID NO.:
- 80, a ligase of SEQ ID NO.:
- 81, a ligase of SEQ ID NO.:
- 82, and combinations thereof;
- (ii) a splint oligonucleotide having a total length of less than 15 nucleotides, wherein said splint oligonucleotide is blocked at its 3′-end with a moiety that prevents polymerase activity;
- (iii) at least one primer;
- and
- (iv) a DNA polymerase,
- c) ligating the oligonucleotides on the first and second probes to one another to produce a target nucleic acid;
- d) amplifying the target nucleic acid;
- and
- e) detecting the amplified target nucleic acid, wherein the ligation and amplification steps occur in a single reaction mixture.
14 more elements
A method for detecting a target in a sample comprising:
- a) binding a first and a second probe, each of which binds specifically to the target, wherein each of the probes comprises a DNA oligonucleotide portion or tail;
- b) adding a mixture to the binding reaction of step (a), the mixture comprising:
- (i) a small-footprint DNA ligase (SFL) selected from the group consisting of, a ligase of SEQ ID NO.:
- 77, a ligase of SEQ ID NO.:
- 78, a ligase of SEQ ID NO.:
- 79, a ligase of SEQ ID NO.:
- 80, a ligase of SEQ ID NO.:
- 81, a ligase of SEQ ID NO.:
- 82, and combinations thereof;
- (ii) a splint oligonucleotide having a total length of less than 15 nucleotides, wherein said splint oligonucleotide is blocked at its 3′-end with a moiety that prevents polymerase activity;
- (iii) at least one primer;
- and
- (iv) a DNA polymerase,
- c) ligating the first and second oligonucleotide tails to produce a ligated oligonucleotide template;
- and
- d) performing a polymerase chain reaction (PCR) of the oligonucleotide template across the first and second oligonucleotide to quantify the said template, wherein steps c) and c) are performed in the same reaction mixture.
13 more elements
Dependencies are inferred from the claim text.