Molecular cloning using rolling circle amplification
First Claim
1. 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 second strand of the circular vector is discontinuous, and wherein the first strand in the circular vector is a closed circular strand, (b) amplifying the first strand by rolling circle replication to form tandem sequence DNA, wherein the amplification results in amplification of the nucleic acid molecule in the first strand.
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Abstract
Disclosed are reagents and a method for efficient in vitro molecular cloning of nucleic acid molecules of interest. Because the method is entirely in vitro, it can be automated and scaled-up in ways that are not possible in cell-based molecular cloning. The method involves insertion of a nucleic acid molecule of interest in a linear vector to form a circular vector where one strand is continuous and the other strand is discontinuous. The continuous strand of the circular vector is then amplified by rolling circle replication, amplifying the inserted nucleic acid molecule in the process. The amplification is rapid and efficient since it involves a single, isothermic reaction that replicates the vector sequences exponentially. The amplification process is amenable to automation where multiple reactions are carried out simultaneously in a small area. The amplified nucleic acid can be used for any purpose and in any manner that nucleic acid cloned or amplified by known methods can be used. This includes sequencing, probing, restriction analysis, subcloning, transcription, hybridization or denaturation analysis, further amplified, and storage for future use or analysis.
218 Citations
45 Claims
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1. 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 second strand of the circular vector is discontinuous, and wherein the first strand in the circular vector is a closed circular strand, (b) amplifying the first strand by rolling circle replication to form tandem sequence DNA, wherein the amplification results in amplification of the nucleic acid molecule in the first strand.
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42. 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.
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45. 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.
Specification