US 2003/0152932 A1 · Detection method using dissociated rolling circle amplification

Abstract

Disclosed are compositions and methods for detecting small quantities of analytes such as proteins and peptides. The method involves associating a DNA circle with the analyte and subsequent release and rolling circle replication of the circular DNA molecule. In the method, an amplification target circle is associated with analytes using a conjugate of the circle and a specific binding molecule that is specific for the analyte to be detected. Amplification target circles not associated with the proteins are removed, the amplification target circles that are associated with the proteins are decoupled from the specific binding molecule and amplified by rolling circle amplification. The amplification is isothermic and can result in the production of a large amount of nucleic acid from each primer. The amplified DNA serves as a readily detectable signal for the analytes.

Claims138

Dependent claims

A method for detecting one or more analytes, the method comprising

(a) bringing into contact one or more analyte samples and one or more reporter binding molecules, wherein each reporter binding molecule comprises a specific binding molecule and an amplification target circle, wherein each specific binding molecule interacts with an analyte directly or indirectly,
incubating the analyte samples and the reporter binding molecules under conditions that promote interaction of the specific binding molecules and analytes, and
separating the specific binding molecules that interact with the analytes from the specific binding molecules that do not interact with the analytes,
(b) decoupling the amplification target circles from the reporter binding molecules that interact with the analytes,
(c) bringing into contact the amplification target circles and one or more rolling circle replication primers, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and
incubating the rolling circle replication primers and amplification target circles under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers,
(d) incubating the rolling circle replication primers and amplification target circles under conditions that promote replication of the amplification target circles,
wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein detection of tandem sequence DNA indicates the presence of the corresponding analytes.

5 more elements

A method for detecting one or more analytes, the method comprising

(a) bringing into contact one or more analyte samples and one or more analyte capture agents, wherein each analyte capture agent interacts with an analyte directly or indirectly, wherein at least one analyte, if present in the analyte sample, interacts with at least one analyte capture agent,
incubating the analyte samples and the analyte capture agents under conditions that promote interaction of the analyte capture agents and analytes,
(b) bringing into contact at least one of the analyte samples and one or more reporter binding molecules, wherein each reporter binding molecule comprises a specific binding molecule and an amplification target circle, wherein each specific binding molecule interacts with an analyte capture agent directly or indirectly,
incubating the analyte samples and the reporter binding molecules under conditions that promote interaction of the specific binding molecules and analyte capture agents, and
separating the specific binding molecules that interact with the analyte capture agents from the specific binding molecules that do not interact with the analyte capture agents,
(c) decoupling the amplification target circles from the reporter binding molecules that interact with the analyte capture agents,
(d) bringing into contact the amplification target circles and one or more rolling circle replication primers, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and
incubating the rolling circle replication primers and amplification target circles under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers,
(e) incubating the rolling circle replication primers and amplification target circles under conditions that promote replication of the amplification target circles,
wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein detection of tandem sequence DNA indicates the presence of the corresponding analytes.

7 more elements

A method for detecting one or more analytes, the method comprising

(a) treating one or more analyte samples so that one or more analytes are modified,
(b) bringing into contact at least one of the analyte samples and one or more reporter binding molecules, wherein each reporter binding molecule comprises a specific binding molecule and an amplification target circle, wherein each specific binding molecule interacts with a modified analyte directly or indirectly,
incubating the analyte samples and the reporter binding molecules under conditions that promote interaction of the specific binding molecules and modified analytes, and
separating the specific binding molecules that interact with the modified analytes from the specific binding molecules that do not interact with the modified analytes,
(c) decoupling the amplification target circles from the reporter binding molecules that interact with the modified analytes,
(d) bringing into contact the amplification target circles and one or more rolling circle replication primers, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and
incubating the rolling circle replication primers and amplification target circles under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers,
(e) incubating the rolling circle replication primers and amplification target circles under conditions that promote replication of the amplification target circles,
wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein detection of tandem sequence DNA indicates the presence of the corresponding modified analytes.

6 more elements

A method for detecting one or more analytes, the method comprising

(a) bringing into contact one or more analyte samples and a set of analyte capture agents, a set of accessory molecules, or both, wherein each analyte capture agent interacts with an analyte directly or indirectly,
(b) prior to, simultaneous with, or following step (a), bringing into contact at least one of the analyte samples and one or more reporter binding molecules, wherein each reporter binding molecule comprises a specific binding molecule and an amplification target circle, wherein each specific binding molecule interacts with an analyte directly or indirectly, wherein each accessory molecule affects the interaction of at least one of the analytes and at least one of the specific binding molecules or at least one of the analyte capture agents,
(c) simultaneous with, or following, steps (a) and (b), incubating the analyte samples, the analyte capture agents, the accessory molecules, and the reporter binding molecules under conditions that promote interaction of the specific binding molecules, analytes, analyte capture agents, and accessory molecules, and
separating the specific binding molecules that interact with the analytes from the specific binding molecules that do no t interact with the analytes,
decoupling the amplification target circles from the reporter binding molecules that interact with the analytes,
(d) bringing into contact the amplification target circles and one or more rolling circle replication primers, wherein the amplification target circles each comprise a single-stranded, circular DNA molecule comprising a primer complement portion, wherein the primer complement portion is complementary to at least one of the rolling circle replication primers, and
incubating the rolling circle replication primers and amplification target circles under conditions that promote hybridization between the amplification target circles and the rolling circle replication primers,
(e) incubating the reporter binding molecules and amplification target circles under conditions that promote replication of the amplification target circles,
wherein replication of the amplification target circles results in the formation of tandem sequence DNA, wherein detection of tandem sequence DNA indicates the presence of the corresponding analytes.

6 more elements

Dependencies are inferred from the claim text.

Detection method using dissociated rolling circle amplification

Life of this patent

Detection method using dissociated rolling circle amplification

Life of this patent

Abstract

Claims138