Methods for array assembly and detection involving elongation of self-complementary looped probes
View Patent ↗Disclosed herein are methods for array assembly and detection. The methods can use an incubation chamber containing a suspension of nucleic acid targets, polymerase and a set of oligonucleotide probes bound to magnetic beads in a randomly dispersed state. Each probe can have a target binding domain that is complementary to a target nucleic acid, a closing domain with a sequence that is complementary to the sequence of the target binding domain, and a joining region between the binding domain and the closing domain, which is not complementary to the target nucleic acid. Method steps can include providing the incubation chamber, placing the incubation chamber in a magnetic trap, generating a magnetic field that induces the magnetic beads to migrate towards a substrate and, once in proximity to the substrate, to interact with each other repulsively and reorganize into arrays, and imaging the array.
1. A method for array assembly and detection comprising:
providing an incubation chamber;
wherein the incubation chamber contains a suspension comprising nucleic acid targets, polymerase and a set of oligonucleotide probes bound to magnetic beads in a randomly dispersed state;
wherein each probe comprises a target binding domain complementary to a target nucleic acid; a closing domain with a sequence that is complementary to the sequence of the target binding domain; and a joining region between the binding domain and the closing domain, which is not complementary to the target nucleic acid;
placing the incubation chamber in a magnetic trap;
generating a magnetic field, wherein the magnetic field induces the magnetic beads in suspension to migrate towards a substrate and, once in proximity to the substrate, the beads interact with each other repulsively and reorganize into arrays; and
imaging the array.
2. The method of claim 1 , wherein the magnetic trap comprises a bottom actuation element and a top element.
3. The method of claim 2 , wherein the incubation chamber is placed in the top element.
4. The method of claim 2 , wherein the magnetic trap further comprises a magnetic core, coil and high permeability layers.
5. The method of claim 1 , wherein the nucleic acid targets are DNA or RNA.
6. The method of claim 1 , wherein the oligonucleotide probes are DNA or RNA.
7. The method of claim 1 , wherein the set of probes comprises at least two probes that differ in the sequence of their target binding domains.
8. The method of claim 1 , wherein the probes are bound to a magnetic bead via the joining region.
9. The method of claim 1 , further comprising placing the set of probes in contact with nucleic acid targets under conditions suitable for capture of the target by a probe and formation of a probe-target complex.
10. The method of claim 9 , wherein the presence of probe-target complexes is detected in real time.
11. The method of claim 9 , further comprising generating conditions suitable for enzyme-mediated probe elongation at the 3′ terminal end if the nucleotide in the target sequence aligned with the 3′ terminal end of the probe is complementary.
12. The method of claim 11 , wherein labeled dNTPs or ddNTPs are incorporated into the elongated probe.
13. The method of claim 12 , further comprising detecting probe-target-associated fluorescence by conducting a thermal stability analysis.
14. The method of claim 13 , wherein the analysis is performed by cycling to a temperature above de-annealing temperature of non-elongated duplexes and then monitoring probe fluorescence to determine probe-target-associated fluorescence.
15. The method of claim 12 , wherein the presence of probe-target complexes is detected in real time by monitoring probe fluorescence from the target-associated state of the probe and comparing it to the pre-assay signal.
16. The method of claim 11 , wherein a reaction time is selected based on the stringency of the imposed conditions, the stringency determining the probability of random formation of a probe-target-enzyme-substrate intermediate state in the formation of elongation product, such that a sufficient number of stable elongated duplex states are capable of being formed.
17. The method of claim 1 , wherein more than one type of oligonucleotide probe is bound to each magnetic bead.
18. The method of claim 1 , wherein a bead map is generated indicating the location and amount of target bound to a probe.