# US 9,752,180 B2 · Methods and compositions for amplification and detection of MicroRNAs

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- Number: US 9,752,180 B2
- Document ID: US9752180B2
- Status: Granted
- Kind: B2
- Application number: 14/076,513
- Filed: 2013-11-11
- Published: 2014-08-07
- Granted: 2017-09-05
- Earliest claimed: 2006-08-25
- Assignees: The Research Foundation for Mental Hygiene, Inc.
- Applicants: Research Foundation for Mental Hygiene, Inc
- Inventors: Stephen D. Ginsberg; Shaoli Che
- Web page: https://patentgrep.com/patent/US9752180B2

## CPC

- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12N 2310/141: MicroRNAs, miRNAs
- C12N 2330/30: chemically synthesised
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

A method and system for amplifying non-coding RNA, microRNA, and small polynucleotide sequences through the generation of a pool of signature sequences to the target sequences. The target sequences can be amplified through DNA synthesis, RNA synthesis, or the combination of DNA and RNA synthesis. The amplification of signature sequences provides an efficient and reproducible mechanism to determine the presence or absence of the target miRNAs, to analyze the quantities of the miRNAs, and for miRNA profiling. The method may also be used for screening for unknown non-coding RNAs, including novel miRNAs.

## Claims

### Claim 1

A method of screening a plurality of unknown microRNA sequences, comprising the steps of:
incubating an RNA sample including the unknown microRNA sequences with a plurality of first oligonucleotides to hybridize the unknown microRNA sequences to the first oligonucleotides;
extending the hybridized unknown microRNA sequences and first oligonucleotides to generate double stranded RNA/DNA hybrid molecules;
cleaving the double stranded RNA/DNA hybrid molecules with a ribonuclease to create overhangs at the 3′ ends that are at least partially complementary to the unknown microRNA sequences;
incubating the cleaved double stranded RNA/DNA molecules with a plurality of second oligonucleotides to hybridize the second oligonucleotide to the overhangs at the 3′ end of the cleaved double stranded RNA/DNA hybrid molecules and form complexes; and
extending the 3′ ends of the first and second oligonucleotides of the complexes to generate double stranded signature sequences; and
confirming the identity of the unknown microRNA sequences.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first oligonucleotides have a random sequence at 3′ end.

### Claim 3 (depends on 2)

The method of claim 2, wherein the second oligonucleotide has a random sequence of 1-25 nucleotides at its 3′ end.

### Claim 4 (depends on 2)

The method of claim 2, wherein the step of confirming the identity of the unknown microRNA sequences comprises cloning and sequencing the unknown microRNA sequences.

### Claim 5 (depends on 2)

The method of claim 2, further comprising the step of removing any free first oligonucleotides before incubating the cleaved double stranded RNA/DNA hybrid molecules with the second oligonucleotides.

### Claim 6 (depends on 2)

The method of claim 2, further comprising the step of removing any free second oligonucleotides before microRNA identification.