Figure 2.
Analysis of the minicircle preparation. (a) Preparation of 86 bp minicircles by LAMA. The lanes of denaturing polyacrylamide gel show single-stranded linear (86 nt L) and circular (86 and 172 nt C), as well as denatured covalently closed double-stranded minicircles (86 and 172 bp C) accumulation after various numbers of cycles. After seven cycles the mixture was treated by exonucleases I and III (lane 6). (b) Confirmation of the circular structure of the 86 bp ligation product by restriction and nicking endonucleases. Samples were separated in denaturing polyacrylamide gel with 10 bp linear DNA marker M (Invitrogen). Lane 1, 86 bp linear DNA; lane 2, 86 bp DNA minicircle; lanes 3–5, 86 bp minicircle treated with single or double digestion by EcoRV and StuI, whose restriction cleavage sites are 43 bp apart by design; lane 6, 86 bp minicircle singly nicked by Nt.BstNBI that runs as a single-stranded circle and a single-stranded linear DNA in denaturing conditions; lane 7, singly-nicked 86 p minicircle treated by Exonuclease I and Exonuclease III which leave only the single-stranded circular DNA.