# US 12,601,008 B2 · Using hairpin formation to identify DNA and RNA sequences having a target nucleic acid sequence

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- Number: US 12,601,008 B2
- Document ID: US12601008B2
- Status: Granted
- Kind: B2
- Application number: 17/980,960
- Filed: 2022-11-04
- Published: 2023-11-02
- Granted: 2026-04-14
- Earliest claimed: 2021-11-04
- Assignees: The Florida State University Research Foundation, Inc.
- Applicants: Florida State University Research Foundation, Inc.
- Inventors: Alan R. Lemmon
- Web page: https://patentgrep.com/patent/US12601008B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6855: Ligating adaptors
- C12Q 1/6844: Nucleic acid amplification reactions
- 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

## Abstract

DNA or RNA including a target nucleotide sequence are amplified by placing a probe nucleotide sequence on the DNA or RNA. The probe nucleotide sequence is complementary to the target nucleotide sequence. The interaction between the probe nucleotide sequence and target nucleotide sequence causes the single-stranded DNA to form a hairpin. The hairpinned single-stranded DNA are selected for amplification.

## Claims

### Claim 1

A method comprising:
ligating a partially double-stranded adapter nucleotide sequence comprising (i) a common adapter sequence and (ii) a probe nucleotide sequence complementary to a target region nucleotide sequence to a target DNA sequence or target RNA sequence comprising the target region nucleotide sequence to form a ligated nucleotide sequence;
denaturing the ligated nucleotide sequence to separate the ligated nucleotide sequence into a single-stranded nucleotide sequence;
cooling the single-stranded nucleotide sequence to allow the single-stranded nucleotide sequence to form a hairpin region when the single-stranded nucleotide sequence includes the target region nucleotide sequence;
digesting the single-stranded nucleotide sequence to remove the adapter nucleotide sequence if the single-stranded nucleotide sequence does not include the target region nucleotide sequence; and
amplifying the single-stranded nucleotide sequence with the formed hairpin region by hybridizing a common primer on the single-stranded nucleotide sequence with the formed hairpin region and extending the common primer.

### Claim 2 (depends on 1)

The method of claim 1, wherein amplifying is achieved by a polymerase chain reaction.

### Claim 3 (depends on 1)

The method of claim 1, wherein the common primer is Common Primer A.

### Claim 4 (depends on 1)

The method of claim 1, wherein the common adapter sequence is Common Adapter A.

### Claim 5 (depends on 1)

The method of claim 1, wherein amplifying includes priming the single-stranded nucleotide sequence with Common Primer A and Common Primer B.

### Claim 6 (depends on 1)

The method of claim 1, wherein:
in the cooling step, the single-stranded nucleotide sequence does not form the hairpin region when the single-stranded nucleotide sequence does not include the target region nucleotide sequence; and
in the digesting step, the adapter nucleotide sequence is not removed from the single-stranded nucleotide sequence when the single-stranded nucleotide sequence includes the target region nucleotide sequence because the formed hairpin region protects the adapter nucleotide sequence.

### Claim 7

A method comprising:
selectively amplifying a target DNA sequence or target RNA sequence containing a pre-determined target region nucleotide sequence therein by:
(a) ligating a partially double-stranded adapter nucleotide sequence comprising (i) a common adapter sequence and (ii) a probe nucleotide sequence complementary to the target region nucleotide sequence to the target DNA sequence or RNA sequence to form a ligated nucleotide sequence;
(b) denaturing the ligated nucleotide sequence to separate the ligated nucleotide sequence into a single-stranded nucleotide sequence;
(c) cooling the single-stranded nucleotide sequence from (b), wherein a hairpin region forms therein when the target region nucleotide sequence is present due to the probe nucleotide sequence bonding to the target region nucleotide sequence, and the hairpin region does not form therein when the target region nucleotide sequence is absent;
(d) digesting the single-stranded nucleotide sequence from (c), wherein the adapter nucleotide sequence is removed therefrom when the target region nucleotide sequence is absent, and the adapter nucleotide sequence is not removed therefrom when the hairpin region formed in (c); and
(e) amplifying the single-stranded nucleotide sequence from (d) for which the hairpin region formed in (b) and the adapter nucleotide sequence was not removed in (d) by hybridizing a common primer thereon and extending the common primer.