Systems and methods for multiplex analysis of PCR in real time
First Claim
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1. A method for quantifying an amount of a nucleic acid target in a sample, comprising:
- (a) combining in a chamber a sample comprising the nucleic acid target, a primer pair for priming amplification of the nucleic acid target, a labeling agent, and a probe complementary to the nucleic acid target, wherein the probe is immobilized on a magnetic bead;
(b) performing an amplification cycle to form amplification products for the nucleic acid target amplified with the primer pair;
(c) hybridizing the amplification products to the probe set;
(d) applying a magnetic field to a surface of the chamber to draw the magnetic bead, the amplification products hybridized to the probe immobilized on the magnetic bead, and any of the labeling agent associated with the amplification product to the surface of the chamber;
(e) detecting a signal from the labeling agent;
(f) removing the magnetic field from the surface of the chamber prior to performing a further amplification cycle;
(g) repeating steps (b) through (f) at least until the signal is distinguishable from background noise; and
(h) quantifying the amount of the nucleic acid target in the sample by correlating the signal with the amount of the nucleic acid target in the sample.
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Abstract
The present invention provides methods and systems for real-time measurements of PCR with multiplexing capability. Certain embodiments relate to methods and systems that use fluorescently encoded superparamagnetic microspheres for the immobilization of amplification products during the PCR process, and an imaging chamber of a measurement device that is also capable of controllable thermal cycling for assisting the PCR process.
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29 Claims
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1. A method for quantifying an amount of a nucleic acid target in a sample, comprising:
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(a) combining in a chamber a sample comprising the nucleic acid target, a primer pair for priming amplification of the nucleic acid target, a labeling agent, and a probe complementary to the nucleic acid target, wherein the probe is immobilized on a magnetic bead; (b) performing an amplification cycle to form amplification products for the nucleic acid target amplified with the primer pair; (c) hybridizing the amplification products to the probe set; (d) applying a magnetic field to a surface of the chamber to draw the magnetic bead, the amplification products hybridized to the probe immobilized on the magnetic bead, and any of the labeling agent associated with the amplification product to the surface of the chamber; (e) detecting a signal from the labeling agent; (f) removing the magnetic field from the surface of the chamber prior to performing a further amplification cycle; (g) repeating steps (b) through (f) at least until the signal is distinguishable from background noise; and (h) quantifying the amount of the nucleic acid target in the sample by correlating the signal with the amount of the nucleic acid target in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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