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1. (WO2017035090) DUMBBELL-PCR: A METHOD TO QUANTIFY SPECIFIC SMALL RNA VARIANTS WITH A SINGLE NUCLEOTIDE RESOLUTION AT TERMINAL SEQUENCES
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Pub. No.: WO/2017/035090 International Application No.: PCT/US2016/048075
Publication Date: 02.03.2017 International Filing Date: 22.08.2016
IPC:
C12Q 1/68 (2006.01) ,C12N 15/09 (2006.01) ,C12N 15/10 (2006.01) ,C12N 15/113 (2010.01) ,A61K 31/7115 (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
68
involving nucleic acids
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09
Recombinant DNA-technology
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09
Recombinant DNA-technology
10
Processes for the isolation, preparation or purification of DNA or RNA
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09
Recombinant DNA-technology
11
DNA or RNA fragments; Modified forms thereof
113
Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides
A HUMAN NECESSITIES
61
MEDICAL OR VETERINARY SCIENCE; HYGIENE
K
PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
31
Medicinal preparations containing organic active ingredients
70
Carbohydrates; Sugars; Derivatives thereof
7088
Compounds having three or more nucleosides or nucleotides
7115
Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
Applicants:
THOMAS JEFFERSON UNIVERSITY [US/US]; 901 Walnut Street Innovation Pillar Philadelphia, Pennsylvania 19107, US
Inventors:
KIRINO, Yohei; US
HONDA, Shozo; US
Agent:
VOS STRACHE, Kyle; US
Priority Data:
62/208,18321.08.2015US
Title (EN) DUMBBELL-PCR: A METHOD TO QUANTIFY SPECIFIC SMALL RNA VARIANTS WITH A SINGLE NUCLEOTIDE RESOLUTION AT TERMINAL SEQUENCES
(FR) PCR DUMBBELL : UN PROCÉDÉ POUR QUANTIFIER DES VARIANTS SPÉCIFIQUES DE PETIT ARN PRÉSENTANT UNE RÉSOLUTION NUCLÉOTIDIQUE UNIQUE AU NIVEAU DES SÉQUENCES TERMINALES
Abstract:
(EN) A method for specifically and efficiently quantify the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5'-Dbs-adapter to the 5'-end of target RNAs, wherein the 5'-Dbs-adapter has a stem-loop structure whose protruding 5'-end base-pairs 5'-end of target RNAs, and wherein the loop region of 5'-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3'-Db-adapter to the 3'-end of target RNAs, wherein the 3'-Db-adapter has a stem-loop structure whose protruding 3'-end base-pairs 3'-end of target RNAs; ligating the both adapters with target RNAs by Rnl2 ligation to form "dumbbell-like" structure; and, amplifying and quantifying the ligation product by TaqMan RT-PCR.
(FR) L'invention concerne un procédé pour quantifier spécifiquement et efficacement l'expression de variants ciblés d'ARN présentant des séquences terminales spécifiques, approprié pour identifier de multiples isoformes portant une hétérogénéité complexe dans les séquences terminales par hybridation d'un adaptateur 5'-Dbs à l'extrémité 5' des ARN cibles, l'adaptateur 5'-Dbs présentant une structure de type tige-boucle dont les paires de bases en saillie de l'extrémité 5' terminent l'extrémité 5' d'ARN cibles et la région de boucle de l'adaptateur 5'-Dbs contenant un espaceur dont une base est absente qui terminera la transcription inverse dans une étape ultérieure ; hybridation d'un adaptateur 3'-Db à l'extrémité 3' des ARN cibles, l'adaptateur 3'-Db présentant une structure de type tige-boucle dont les paires de bases en saillie de l'extrémité 3' terminent l'extrémité 3' d'ARN cibles ; ligature des deux adaptateurs aux ARN cibles par une ligature Rnl2 pour former une structure de type "dumbbell" (haltère) ; et amplification et quantification du produit de ligature par RT-PCR TaqMan.
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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, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 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)