US 12,442,036 B2
Multiplex assay for nucleic acid detection
Yuan-Jyue Chen, Seattle, WA (US); Karin Strauss, Seattle, WA (US); and Bichlien H Nguyen, Seattle, WA (US)
Assigned to Microsoft Technology Licensing, LLC, Redmond, WA (US)
Filed by Microsoft Technology Licensing, LLC, Redmond, WA (US)
Filed on Nov. 29, 2021, as Appl. No. 17/456,855.
Prior Publication US 2023/0167485 A1, Jun. 1, 2023
Int. Cl. C12Q 1/6816 (2018.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01)
CPC C12Q 1/6816 (2013.01) [C12N 15/11 (2013.01); C12N 9/222 (2025.05)] 20 Claims
OG exemplary drawing
 
1. A multiplex assay system for deoxyribonucleic acid (DNA) detection, the assay comprising:
a substrate having a plurality of physically separated assay locations, each assay location of the plurality of assay locations including:
a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 12 (Cas12) enzyme that cleaves single-stranded deoxyribonucleic acid (ssDNA),
a guide ribonucleic acid (gRNA) that forms a complex with the Cas12 enzyme, a sequence of the gRNA being complementary to a sequence of a target DNA, and
a quencher-labeled oligonucleotide, the quencher-labeled oligonucleotide being an ssDNA molecule and having an anchor portion configured to bind to the substrate, and a quencher configured to absorb energy emitted by an adjacent fluorophore;
a sample, a portion of which is distributed to each assay location of the plurality of assay locations; and
a fluorophore-labeled oligonucleotide that is added to each assay location of the plurality of assay locations subsequent to a distribution of the sample, the fluorophore-labeled oligonucleotide being an RNA molecule and having a region configured to bind a non-anchor portion of the quencher-labeled oligonucleotide, and a fluorophore configured to emit light upon excitation in an absence of the quencher, wherein
the gRNA is configured to recognize the target DNA in the sample, thereby guiding the Cas12 enzyme to the target DNA and activating the Cas12 enzyme to cleave ssDNA molecules,
the quencher-labeled ssDNA molecule is configured to be cleaved by the activated Cas12 enzyme, thereby resulting in a truncated quencher-labeled ssDNA molecule that lacks the quencher when the target DNA is present in the sample, and
the fluorophore-labeled RNA is configured to not be cleaved by the Cas12 enzyme, thereby permitting identification of the target DNA in the sample via detection of light emitted by the fluorophore at one or more assay locations upon excitation.