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(12) Patent Application Publication (10) Pub. No.: US 2009/0275039 A1 Moser Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2009/0275039 A1 Moser Et Al US 20090275039A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0275039 A1 Moser et al. (43) Pub. Date: Nov. 5, 2009 (54) METHODS FOR DETECTION AND Related U.S. Application Data QUANTIFICATION OFSMALL RNA (60) Provisional application No. 61/046,162, filed on Apr. 18, 2008. (76) Inventors: Michael J. Moser, Madison, WI (US); Vecheslav A. Elagin, Publication Classification Waunakee, WI (US) (51) Int. Cl. CI2O I/68 (2006.01) Correspondence Address: (52) U.S. Cl. ............................................................ 435/6 FOLEY & LARDNER LLP (57) ABSTRACT 150 EAST GILMAN STREET, P.O. BOX 1497 MADISON, WI 53701-1497 (US) Disclosed are methods, kits, and components for detecting small RNA molecules, such as microRNA and siRNA, in a sample. The methods utilize primers and reporter molecules (21) Appl. No.: 12/425,088 comprising non-natural bases. The disclosed kits may include one or more components for performing the disclosed meth (22) Filed: Apr. 16, 2009 ods. Patent Application Publication Nov. 5, 2009 Sheet 1 of 7 US 2009/0275039 A1 I 9I IZ :ONGIÕIGIS :ONCIIÕIGIS :ONOLIÕIGIS :ONCIIÕES I”SOIH 0£:ON 7£:ON 69:ON Zý:ON £7:ON 7:ON G:ON 97:ON CII CII CII CII OI CII CII CII L7:ON 87:ON 67:ON 09:ON IG:ON ZG:ON 9G:ON 7G:ON CII CII CII QII CII CII CII CII Patent Application Publication Nov. 5, 2009 Sheet 2 of 7 US 2009/0275039 A1 F.G. 2 500 -500 D Ct Tm O -1000 et-a 32.8 74.1 l -500 -2000 et-7C NIA N/A -2500 -3COO et-f NA. NIA -3500 let-7c, let-7f -500 sstar' - 1000 60 65 70 75 80 85 9 Temperature (C) Patent Application Publication Nov. 5, 2009 Sheet 3 of 7 US 2009/0275039 A1 FIG 3 500 -500 let-7a, let-7f ID Ci Tm -1000 let-7a NIA N/A -1500 -2000 et-7C 272 76.0 -2500 et-7f N/A NJA .3000 -3500 2O 25 3O 35 40 Cycle -250 -500 -750 60 65 70 75 8O 85 90 Temperature (C) Patent Application Publication Nov. 5, 2009 Sheet 4 of 7 US 2009/0275039 A1 FIG. 4 500 -500 let-7a, let-7c O Ci m -1000 let-7a NAA NIA -1500 -2000 et-7C NA NAA -2500 et-7f 27.0. 73.5 -3000 -3500 20 25 3O 35 40 250 -500 750 60 65 70 75 8O 85 90 emperature (C) Patent Application Publication Nov. 5, 2009 Sheet 5 of 7 US 2009/0275039 A1 F.G. 5 SOC - OOC et-7a 33.5 74.3 - 1500 -2000 et-7C N/A NIA -250 300 et-7f 34.2 73.2 -3500 -500 -1000 60 65 70 75 80 85 90 Temperature (C) Patent Application Publication Nov. 5, 2009 Sheet 6 of 7 US 2009/0275039 A1 FIG. 6A 45 FAM CT 15 4 CD O 3. 3 O 2 30 8 E 25 H O 20 100 fM 100 fM 10 fM 10 fM 1 fM 1 fM let-7C let-7a let-7C let-7a let-7C let-7a F.G. 6B HEX CT 3 O 2 5 2 O 100 fM 100 fM 10 fM 10 fM 1 fM 1 fM let-7C let-7a let-7c let-7a let-7C let-7a Patent Application Publication Nov. 5, 2009 Sheet 7 of 7 US 2009/0275039 A1 F.G. 6C ACT 100 fM 100 fM 10 fM 10 fM 1 fM 1 fM let-7C let-7a let-7C let-7a let-7C let-7a US 2009/0275039 A1 Nov. 5, 2009 METHODS FOR DETECTION AND 0007 Each of the first primer and the second primer may QUANTIFICATION OFSMALL RNA comprise a target binding region and a tail region. In some embodiments, the target binding regions of the first primer CROSS-REFERENCE TO RELATED and/or the second primer comprise from about 7 to about 10 APPLICATIONS nucleotides. Likewise, the tail regions of the first primer and/ 0001. This application claims priority to U.S. Provisional or the second primer each comprise from about 10 to about 25 Application No. 61/046,162, filed Apr. 18, 2008, the entire nucleotides, or from about 7 to about 15 nucleotides. The tail contents of which are hereby incorporated by reference in its region of the first primer, the second primer, or both may entirety. comprise one or more non-natural bases. 0008. The non-natural bases used in the methods TECHNICAL FIELD described herein may be selected from iso-C and iso-G. In suitable embodiments, the first non-natural base is iso-C and 0002 The present methods and kits relate broadly to the the second non-natural base is iso-G. For example, the identification of nucleic acids using nucleic acid amplifica reporter may comprise an nucleotide triphosphate of iso-G tion techniques. In particular, the methods and kits relate to conjugated to a label. Such as a quencher. methods of detecting and quantifying Small RNA molecules, such as microRNA. 0009. The extension of the first primer is under conditions suitable to produce a first reaction product of the small RNA. Typically, these conditions allow for the specific hybridiza BACKGROUND tion of the target binding region of the first primer to the target 0003 MicroRNA (miRNA) are small RNA molecules that Small RNA. In some embodiments, the step of annealing and are expressed as pol II transcripts in eukaryotic organisms. extension to produce a first reaction product is performed at These molecules have been shown to regulate gene expres temperature from about 15° C. to about 50° C. Sion, mRNA splicing, and histone formation. They also have 0010. The step of amplifying the first reaction product been shown to have tissue-specific and developmental-spe with the first primer and the second primer is under conditions cific expression patterns. Thus, these small RNA molecules Suitable to produce an amplification product. In the first round are of interest in the elucidation of biological processes, dis of amplification, these conditions allow for the specific ease states, and development. hybridization of the target binding region of the second primer to the first amplification product. In some embodi SUMMARY ments, this annealing step is performed at a temperature from 0004. There are provided herein methods and kits for about 35°C. to about 45° C. Subsequent annealing steps may quickly, easily and inexpensively detecting and quantifying be performed at a temperature at least about 55° C. small RNA in a biological sample. The methods described 0011. The primers described herein may be designed to herein provide a homogenous, probe-free assay for the detec have varying specificity for related small RNA isoforms. In tion of a variety of small RNAs, including miRNA and Some embodiments, the primers are capable of discriminating siRNA. between related isoforms. In other embodiments, the primers 0005. In one aspect, the invention provides a method com are not capable of discriminating between related isoforms. prising: (a) contacting a sample to be tested for the presence Therefore, in some methods, the first primer, the second or absence of a target small RNA with: (i) a first primer primer, or both are specific for a single small RNA isoform. comprising a target binding region and a tail region, wherein For example, the single small RNA isoform differs from other the target binding region comprises about 12 or fewer nucle isoforms at one or more specific nucleotide positions, and at otides that are complementary to the small RNA; (ii) a second least the 4 terminal nucleotides at the 3' end of first primer, the primer comprising a target binding region and a tail region, second primer, or both are complementary to the single iso wherein the target binding region comprises about 12 or form, but not complementary to the other isoforms. fewer nucleotides that are complementary to the reverse 0012. In some embodiments, one or both of the first primer complement of the small RNA, wherein the tail regions of the and the second primer comprise a first label. In suitable first primer, the second primer, or both the first primer and the embodiments, the first label is a fluorophore. The first label second primer comprise a first label and a first non-natural may interact with the second label, i.e., the first label and the base; (b) extending the first primer under conditions suitable second label may form an interactive dye-quencher pair. In to produce a first reaction product of the small RNA, if present such embodiments, the first label is a fluorophore and the in the sample; (c) amplifying the first reaction product with second label is a quencher. For example, the fluorophore may the first primer and the second primer under conditions Suit be FAM or HEX and the quencher may be Dabcyl. able to produce an amplification product, wherein a second 0013. In some embodiments, the methods may be used to non-natural base conjugated to a second label is incorporated quantitate the amount of target Small RNA in a sample. In into the amplification product opposite the first non-natural Such embodiments, the step of detecting comprises quantify base, and wherein the first non-natural base is iso-C or iso-G ing the amount of the small RNA in the sample. The step of and the second non-natural base is the other of iso-Coriso-G; quantifying may comprise correlating the amount of signal and (d) detecting the amplification products produced in step observed from the first label, the second label or both the first (c) by observing a signal from the first label, the second label, label and the second label, to the amount of small RNA in the or both the first label and the second label.
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