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1. (WO2019067711) DIGITAL NUCLEASE DETECTION COMPOSITIONS AND METHODS
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Pub. No.: WO/2019/067711 International Application No.: PCT/US2018/053107
Publication Date: 04.04.2019 International Filing Date: 27.09.2018
IPC:
C12Q 1/04 (2006.01) ,C12Q 1/68 (2018.01) ,G01N 33/50 (2006.01) ,G01N 33/53 (2006.01) ,G01N 33/543 (2006.01)
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
Q
MEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICRO-ORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
1
Measuring or testing processes involving enzymes or micro-organisms; Compositions therefor; Processes of preparing such compositions
02
involving viable micro-organisms
04
Determining presence or kind of micro-organism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
Q
MEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICRO-ORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
1
Measuring or testing processes involving enzymes or micro-organisms; Compositions therefor; Processes of preparing such compositions
68
involving nucleic acids
G PHYSICS
01
MEASURING; TESTING
N
INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33
Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48
Biological material, e.g. blood, urine; Haemocytometers
50
Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
G PHYSICS
01
MEASURING; TESTING
N
INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33
Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48
Biological material, e.g. blood, urine; Haemocytometers
50
Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
53
Immunoassay; Biospecific binding assay; Materials therefor
G PHYSICS
01
MEASURING; TESTING
N
INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33
Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48
Biological material, e.g. blood, urine; Haemocytometers
50
Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
53
Immunoassay; Biospecific binding assay; Materials therefor
543
with an insoluble carrier for immobilising immunochemicals
Applicants:
UNIVERSITY OF IOWA RESEARCH FOUNDATION [US/US]; 112 N. Capitol Street 6 Gilmore Hall Iowa City, Iowa 52242-5500, US
Inventors:
MCNAMARA, James, O.; US
Agent:
VIKSNINS, Ann, S.; US
HARRIS, Robert, J.; US
MALEN, Peter, L.; US
KRUEGER, Katherine, A.D.; US
MICKELSON, John, W.; US
ANDRUS, Alex; US
Priority Data:
62/565,67429.09.2017US
Title (EN) DIGITAL NUCLEASE DETECTION COMPOSITIONS AND METHODS
(FR) COMPOSITIONS ET PROCÉDÉS NUMÉRIQUES DE DÉTECTION DE NUCLÉASE
Abstract:
(EN) In certain embodiments, the present invention provides a detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide. As used herein, the term "picodroplet" comprises a liquid droplet that has a volume of 0.014 to 2.6 picoliters. In certain embodiments, the present invention provides a method of detecting at least one individual nuclease molecule present in a sample, comprising contacting an aqueous sample suspected of containing at least one nuclease with at least one detection composition comprising a picodroplet comprising (a) an aqueous solution, and (b) a substrate probe comprising (i) an oligonucleotide of 2 to 75 nucleotides in length, (ii) a fluorophore operably linked to the oligonucleotide, and (iii) a quencher operably linked to the oligonucleotide to form an aqueous reaction mixture; emulsifying the aqueous mixture in oil to form picoliter-scale droplets in an emulsion, (c) incubating the picoliter-scale droplets in the emulsion in order for the nuclease, if present, to digest the substrate probes linked to the microbeads; recovering the microbeads; and detecting fluorescence emitting from the microbeads.
(FR) Selon certains modes de réalisation, la présente invention concerne une composition de détection comprenant une picogouttelette comprenant (a) une solution aqueuse, et (b) une sonde à substrat comprenant (i) un oligonucléotide de 2 à 75 nucléotides de long, (ii) un fluorophore fonctionnellement lié à l'oligonucléotide, et (iii) un agent d'extinction fonctionnellement lié à l'oligonucléotide. Tel qu'il est utilisé ici, le terme "picogouttelette" désigne une gouttelette liquide ayant un volume de 0,014 à 2,6 picolitres. Dans certains modes de réalisation, la présente invention concerne un procédé de détection d'au moins une molécule de nucléase individuelle présente dans un échantillon, comprenant la mise en contact d'un échantillon aqueux suspecté de contenir au moins une nucléase avec au moins une composition de détection comprenant une picogouttelette comprenant (a) une solution aqueuse, et (b) une sonde à substrat comprenant (i) un oligonucléotide de 2 à 75 nucléotides de long, (ii) un fluorophore fonctionnellement lié à l'oligonucléotide, et (iii) un agent d'extinction fonctionnellement lié à l'oligonucléotide pour former un mélange réactionnel aqueux ; l'émulsionnement du mélange aqueux dans l'huile pour former des picogouttelettes en émulsion, (c) l'incubation des picogouttelettes en émulsion afin que la nucléase, si présente, digère les sondes à substrat liées aux microbilles ; la récupération des microbilles ; et la détection d'une émission de fluorescence émanant des microbilles.
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Designated States: AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW
African Regional Intellectual Property Organization (ARIPO) (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW)
Eurasian Patent Office (AM, AZ, BY, KG, KZ, RU, TJ, TM)
European Patent Office (EPO) (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR)
African Intellectual Property Organization (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG)
Publication Language: English (EN)
Filing Language: English (EN)