Method for producing circular DNA formed from single-molecule DNA

摘要

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.

主权项

1. A method for producing a circular DNA, the method comprising the following steps:
1) providing a DNA molecule; 2) a step of covalently binding an adapter (A) to one end of the DNA molecule, and covalently binding to the other end of the DNA molecule an adapter (B) comprising an adapter (b) and the adapter (A), wherein:
the adapter (B) binds to the DNA molecule via the adapter (b) and not the adapter (A),the adapter (A) comprises a cleavage site capable of generating a cleavage end which can non-specifically bind to any cleavage end of the adapter (A), andthe adapter (b) comprises a cleavage site capable of generating a cleavage end which can specifically bind only to the cleavage end from the same adapter (b); 3) a first cleavage step of cleaving the DNA molecule obtained in step 2) at the cleavage site of each of the adapters (A); 4) a first circularization step of binding both ends of the DNA molecule obtained in step 3) to circularize the DNA molecule; 5) a step of eliminating uncircularized linear DNA molecules obtained in step 4); 6) a second cleavage step of cleaving the circular DNA molecules from step 5) at the cleavage site of the adapter (b); and 7) a second circularization step of binding both ends of the DNA molecule obtained in step 6) to circularize the DNA molecule and obtain a circular DNA.