# US 9,434,941 B2 · Method for producing circular DNA formed from single-molecule DNA

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- Number: US 9,434,941 B2
- Document ID: US9434941B2
- Status: Granted
- Kind: B2
- Application number: 14/594,458
- Filed: 2015-01-12
- Published: 2015-07-16
- Granted: 2016-09-06
- Earliest claimed: 2010-09-02
- Assignees: Kurume University
- Applicants: Kurume University
- Inventors: Shinichi Mizuno; Koji Nagafuji; Takashi Okamura
- Web page: https://patentgrep.com/patent/US9434941B2

## CPC

- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 2525/307: Circular oligonucleotides
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- 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

There is provided a method for producing a circular DNA which consists of a circular DNA formed from a single-molecule DNA and which does not comprise circular DNA formed from multiple-molecule DNA. According to the method of the present invention, a circular DNA molecule formed only from a single-molecule DNA can be reliably produced.

## Claims

### Claim 1

An adapter for producing a circular DNA, the adapter consisting of two DNA strands complementary to each other having the following structure y1-Y-y2-X:
wherein
X represents a double-stranded DNA being a restriction enzyme recognition site comprising a palindromic sequence;
y1 and y2 represent double-stranded DNAs respectively comprising identical nick-generating enzyme recognition sites being reversely oriented to each other;
Y represents a double-stranded DNA sequence having a unique sequence for each DNA molecule to be circularized, n represents an integer of 4 to 15, N1 to Nn, which may be the same or different, each represent a deoxyribonucleotide selected from the group consisting of dAMP, dCMP, dGMP and dTMP, and N′1 to N′n represent the following deoxyribonucleotides respectively corresponding to the N1 to Nn,
TABLE 1
Nk
N′k
dAMP
dTMP
dCMP
dGMP
dGMP
dCMP
dTMP
dAMP
wherein k represents an integer from 1 to n.

### Claim 2 (depends on 1)

The adapter according to claim 1, wherein the nick-generating enzyme is Nb.BtsI or Nt.BsqQI.

### Claim 3 (depends on 1)

A kit for producing circular DNA, comprising an adapter consisting of a double-stranded DNA comprising a restriction enzyme recognition site identical to the restriction enzyme recognition site represented by X defined in claim 1, and the adapter according to claim 1.