Real-time detection of rolling circle amplification products
First Claim
1. A method of detecting amplification products during multiply-primed rolling circle amplification, the method comprising incubating a mixture comprising an amplification target circle, one or more rolling circle replication primers, and one or more fluorescent change probes, under conditions that promote rolling circle replication of the amplification target circle, wherein the rolling circle replication is primed from a plurality of locations on the amplification target circle, wherein the rolling circle replication results in formation of tandem sequence DNA, and detecting, during the incubation, fluorescent change probes interacting with the tandem sequence DNA.
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Abstract
Disclosed are compositions and methods for real-time detection of rolling circle amplification products. Real-time detection is detection that takes place during the amplification reaction or operation. Real-time detection can be accomplished by, for example, using fluorescent change probes and/or primers during amplification. The fluorescent signals can be proportional to the amount of amplification product. The amplification can be multiply-primed rolling circle amplification in which replication of a circular template is primed at a plurality of sites on the circular template. Multiply-primed RCA increases the sensitivity of singly-primed rolling circle amplification. Multiply-primed RCA can be performed using a single primer (which hybridizes to multiple sites on the amplification target circle) or multiple primers (each of which can hybridize to a single site on the amplification target circle or multiple sites on the amplification target circle). Fluorescent change probes and primers are probes and primers that involve a change in fluorescence intensity or wavelength based on a change in the form or conformation of the probe or primer and nucleic acid to be detected, assayed or replicated.
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Citations
83 Claims
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1. A method of detecting amplification products during multiply-primed rolling circle amplification, the method comprising
incubating a mixture comprising an amplification target circle, one or more rolling circle replication primers, and one or more fluorescent change probes, under conditions that promote rolling circle replication of the amplification target circle, wherein the rolling circle replication is primed from a plurality of locations on the amplification target circle, wherein the rolling circle replication results in formation of tandem sequence DNA, and detecting, during the incubation, fluorescent change probes interacting with the tandem sequence DNA.
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74. A method of detecting amplification products during multiply-primed rolling circle amplification, the method comprising
incubating a mixture comprising an amplification target circle and one or more rolling circle replication primers under conditions that promote rolling circle replication of the amplification target circle, wherein one or more of the rolling circle replication primers comprise a fluorescent change primer, wherein the rolling circle replication is primed from a plurality of locations on the amplification target circle, wherein the rolling circle replication results in formation of tandem sequence DNA, and detecting, during the incubation, fluorescent change primers incorporated into the tandem sequence DNA.
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76. A method of detecting amplification products during multiply-primed rolling circle amplification, the method comprising
incubating a mixture comprising an amplification target circle, one or more rolling circle replication primers, and one or more DNA strand displacement primers under conditions that promote rolling circle replication of the amplification target circle, wherein one or more of the DNA strand displacement primers comprise a fluorescent change primer, wherein the rolling circle replication is primed from a plurality of locations on the amplification target circle, wherein the rolling circle replication results in formation of tandem sequence DNA, and detecting, during the incubation, fluorescent change primers incorporated into the tandem sequence DNA.
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78. A method of detecting amplification products during multiply-primed rolling circle amplification, the method comprising
incubating a mixture comprising an amplification target circle, one or more rolling circle replication primers, and one or more fluorescent change probes, under conditions that promote rolling circle replication of the amplification target circle, wherein one or more of the rolling circle replication primers comprise a fluorescent change primer, wherein the rolling circle replication is primed from a plurality of locations on the amplification target circle, wherein the rolling circle replication results in formation of tandem sequence DNA, and detecting, during the incubation, fluorescent change probes interacting with the tandem sequence DNA, fluorescent change primers incorporated into the tandem sequence DNA, or both.
Specification