Highly specific circular proximity ligation assay
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsHenrik H.J. PERSSON, Roxana JALILI, Joseph L. HORECKA, Ronald W. DAVIS
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Abstract
Provided herein is a circular proximity ligation assay in which proximity-probes are employed as bridges to connect two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. The addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation may have selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR.
Claims14
1-20. (canceled)
A method for sample analysis comprising:
- (a) incubating a sample comprising a target analyte that is at a concentration of less than 100 nM with:
- (i) a first conjugate comprising a binding agent and a first splint oligonucleotide, and
- (ii) a second conjugate comprising a binding agent and a second splint oligonucleotide,
- wherein the first and second conjugates are one or more proteins that have been split into a first portion and a second portion and then conjugated to the first and second splint oligonucleotides,
- under conditions suitable for binding of the binding agents of the first and second conjugates to the target analyte, to produce a product;
- (b) incubating at least some of the product of step (a) with:
- (i) a set of probes that produces a ligatable circle only when the probes are hybridized to the first and second splint oligonucleotides;
- and
- (ii) a ligase;
- to produce a reaction mix comprising covalently closed circular molecules;
- (c) terminating the ligation of step (b);
- and
- (d) after step (c), quantifying the amount of covalently closed circular molecules produced in step (b).
Dependencies are inferred from the claim text.