Methods for preparing nucleic acid molecules for sequencing

Abstract

Means and methods for preparing double stranded target DNA molecules for sequencing. In embodiments double stranded backbone DNA molecules comprising 5' and 3′ ends are provided that are: ligation compatible with 5′ and 3′ ends of the target DNA; form a first restriction enzyme recognition site when self-ligated; in a form that enables self-ligation. Methods may comprise providing, if not already present, the target DNA with 5′ and 3′ ends that are in a form that prevents self-ligation and that are ligation compatible with the backbone DNA 5′ and 3′ ends. Methods may further comprise ligating the target DNA to the backbone DNA in the presence of a ligase and a first restriction enzyme that cuts the first restriction enzyme recognition site, thereby producing at least one DNA circle comprising a backbone DNA molecule and a target DNA molecule. Linear DNA may be removed at this time and subsequently a concatemer DNA molecule comprising an ordered array of copies of the at least one DNA circle through rolling circle amplification is produced that can be sequenced.