公开(公告)号：US10301672B2

公开(公告)日：2019-05-28

申请号：US15527560

申请日：2015-11-18

Applicant: Japan Science and Technology Agency

Inventor： Masayuki Su'etsugu ,  Hiroko Kobayashi

IPC: C12Q1/68 ,  C12P19/34 ,  C12N15/00 ,  C12Q1/6844 ,  C12N9/00 ,  C12N9/12 ,  C12N9/16 ,  C12N9/90 ,  C12N15/09 ,  C12Q1/6853 ,  C12Q1/686

Abstract: The present invention provides a method for easily and exponentially amplifying circular DNA, particularly long chain circular DNA, in a cell-free system. Specifically, the present invention provides a method for amplifying circular DNA in which circular DNA having a replication origin sequence (origin of chromosome (oriC)) is mixed with a reaction solution containing the following enzyme groups to form a reaction mixture, which is then reacted under an isothermal condition, the enzyme groups being: (1) a first enzyme group that catalyzes replication of circular DNA; (2) a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane; and (3) a third enzyme group that catalyzes a separation of two sister circular DNAs.

公开(公告)号：US20190276883A1

公开(公告)日：2019-09-12

申请号：US16302485

申请日：2017-05-17

Applicant: Japan Science and Technology Agency

Inventor： Masayuki Su'etsugu ,  Hiroko Tsujimoto ,  Takeshi Shinohara

IPC: C12Q1/6855

Abstract: Provided is a method capable of simply and exponentially amplifying circular DNA, and particularly, long-chain circular DNA, in a cell-free system. Specifically, provided herein is a method for amplifying circular DNA which comprises mixing circular DNA having a replication origin sequence (origin of chromosome (oriC)) with a reaction solution comprising: a first enzyme group that catalyzes replication of circular DNA; a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane; a third enzyme group that catalyzes a separation of two sister circular DNAs; and also, a buffer, NTP, dNTP, a magnesium ion source, and an alkali metal ion source, to form a reaction mixture, which is then reacted.

公开(公告)号：US20190249236A1

公开(公告)日：2019-08-15

申请号：US16371766

申请日：2019-04-01

Applicant: Japan Science and Technology Agency

Inventor： Masayuki Su'etsugu ,  Hiroko Kobayashi

IPC: C12Q1/6844 ,  C12Q1/686 ,  C12Q1/6853 ,  C12P19/34 ,  C12N15/09 ,  C12N9/00 ,  C12N9/90 ,  C12N9/16 ,  C12N9/12

CPC classification number: C12Q1/6846 ,  C12N9/00 ,  C12N9/12 ,  C12N9/16 ,  C12N9/90 ,  C12N15/00 ,  C12N15/09 ,  C12P19/34 ,  C12Q1/6853 ,  C12Q1/686

Abstract: The present invention provides a method for easily and exponentially amplifying circular DNA, particularly long chain circular DNA, in a cell-free system. Specifically, the present invention provides a method for amplifying circular DNA in which circular DNA having a replication origin sequence (origin of chromosome (oriC)) is mixed with a reaction solution containing the following enzyme groups to form a reaction mixture, which is then reacted under an isothermal condition, the enzyme groups being:(1) a first enzyme group that catalyzes replication of circular DNA; (2) a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane and (3) a third enzyme group that catalyzes a separation of two sister circular DNAs.