Molecular cloning using rolling circle amplification

A method of isolating and amplifying nucleic acid molecules, the method comprising

• (a) ligating a plurality of nucleic acid molecules into a plurality of linear vectors in a single reaction to form a plurality of circular vectors, each circular vector comprising a vector and a nucleic acid molecule,
• wherein the linear vectors are double-stranded linear nucleic acid comprising two nucleic acid strands, wherein the circular vectors each contain at least one nick, gap, or overlap in the second strand, and wherein the first strand in each circular vector is a closed circular strand,
• (b) separating the first strands from the second strands,
• (c) diluting and dividing the first strands to produce a plurality of separate amplification reactions that, on average, each contain a single circular vector,
• (d) amplifying the first strands of the plurality of circular vectors by rolling circle replication to form tandem sequence DNA,
• wherein the amplification results in amplification of the nucleic acid molecules in the first strands.

A method of isolating and amplifying a nucleic acid molecule, the method comprising

• (a) ligating a nucleic acid molecule into a linear vector to form a circular vector comprising the vector and the nucleic acid molecule,
• wherein the linear vector is a double-stranded linear nucleic acid comprising two nucleic acid strands, wherein the circular vector contains at least one nick in the second strand and wherein the first strand in the circular vector is a closed circular strand,
• (b) amplifying the first strand,
• wherein the amplification results in amplification of the nucleic acid molecule in the first strand.