Patent SG-11201907762R-A (SG)
Title: Methods for multiplex detection of molecules
Inventor: SCOLNICK JONATHAN
IPC: G01N33/50
Filed: 2018-03-23
Abstract: INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date 27 September 2018 (27.09.2018) WIPO I PCT ~~ll~~~~~~~~ 011101010VIIIOH olo 010010IH001010m 1111101 ow (10) International Publication Number WO 2018/174827 Al (51) International Patent Classification: C12Q 1/6804 (2018.01) G01N 33/50 (2006.01) (21) International Application Number: PCT/SG2018/050130 (22) International Filing Date: 23 March 2018 (23.03.2018) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 10201702423X 24 March 2017 (24.03.2017) SG (71) Applicants: NATIONAL UNIVERSITY OF SINGA- PORE [SG/SG]; 21 Lower Kent Ridge Road, Singapore 119077 (SG). AGENCY FOR SCIENCE, TECHNOL- OGY AND RESEARCH [SG/SG]; 1 Fusionopolis Way, #20-10 Connexis, Singapore 138632 (SG). (72) Inventors: SCOLNICK, Jonathan; c/o National Univer- sity of Singapore, Yong Loo Lin School of Medicine, De- partment of Physiology, 21 Lower Kent Ridge Road, Singa- pore 119077 (SG). YEO, Eugene; c/o National University of Singapore, Yong Loo Lin School of Medicine, Depart- ment of Physiology, 21 Lower Kent Ridge Road, Singapore 119077 (SG). HOON, Shawn; Biomedical Sciences Insti- tute, 61 Biopolis Drive, Proteos #03-13, Singapore 138673 (SG). (74) Agent: AMICA LAW LLC; 30 Raffles Place, #14-01 Chevron House, Singapore 048622 (SG). (81) Designated States (unless otherwise indicated, for every kind of national protection available): 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, (54) Title: METHODS FOR MULTIPLEX DETECTION OF MOLECULES General workflow Single cell isolation Eg, 10X chromium, seq- well, Fluidigm Cl, 384 well plates, BD Rhapsody Sequencing Total cDNA amplification and sequencing library preparation Targeted cDNA amplification conjugated oligo from cDNA Conjugate amplification and sequencing Reverse transcription ► Separation of Analysis 4 1-1 N N GC N 00 O 1-1 1-1 N C FIG. 10 Sequence read counts used to determine protein levels (57) : The present invention provides methods, antibodies and kits for detecting and/or quantifying expression of both non- nucleic acid molecules, such as proteins, and nucleic acid molecules in a single sample or single cell. The antibody is covalently conjugated to an oligonucleotide, such as a single-stranded DNA molecule, which comprises an identification tag and a blocking group, such as a ddNTP or an inverted dTTP, which prevents extension of the oligonucleotide by a polymerase. The method comprises the steps of reverse transcribing the conjugated oligonucleotide and the target nucleic acid simultaneously, amplifying both transcription production, and detecting the amplicons thereof. The method is also useful for detecting and/or quantifying the number of cells in a sample expressing a given non -nucleic acid molecule (e.g. protein). [Continued on next page] WO 2018/174827 Al MIDEDIMOMOIDEIREEMOMMEIVEIMERVOIMIE 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. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (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), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Declarations under Rule 4.17: — of inventorship (Rule 4.17 (iv)) Published: with international search report (Art. 21(3)) in black and white; the international application as filed contained color or greyscale and is available for download from PATENTSCOPE