Method and device for detecting the presence of a single target nucleic acid in a sample
First Claim
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1. A method for amplifying a target nucleic acid when present in a sample, the method comprising:
- introducing to sample retaining means a single aqueous sample and a nongaseous fluid that is immiscible with the sample, wherein the sample comprises reagents sufficient to perform a primer-based amplification of a nucleic acid when present in said sample;
segregating the sample into a plurality of aliquots, wherein the aliquots have reaction volumes ranging from 1 picoliter to 1 microliter, and wherein the aliquots are segregated and entrapped by the nongaseous fluid in the sample retaining means; and
subjecting the plurality of aliquots in the sample retaining means to a primer-based amplification reaction whereby reagent primers anneal to the target nucleic acid to amplify the target nucleic acid when said target nucleic acid is present in said sample.
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Abstract
A method comprising subjecting one or more sample portion(s) to a single amplification step, thereby amplifying a single molecule in the sample portion to a detectable level, and, in some embodiments, then determining whether the sample portion contains at least one molecule of the target nucleic acid. In some embodiments, the sample portion is in a porous sample structure, or in a sample chamber which comprises means for minimizing diffusion of the sample portion, or in a sample chamber which is inside a microcapillary device, or in a sample retaining means.
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Citations
7 Claims
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1. A method for amplifying a target nucleic acid when present in a sample, the method comprising:
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introducing to sample retaining means a single aqueous sample and a nongaseous fluid that is immiscible with the sample, wherein the sample comprises reagents sufficient to perform a primer-based amplification of a nucleic acid when present in said sample; segregating the sample into a plurality of aliquots, wherein the aliquots have reaction volumes ranging from 1 picoliter to 1 microliter, and wherein the aliquots are segregated and entrapped by the nongaseous fluid in the sample retaining means; and subjecting the plurality of aliquots in the sample retaining means to a primer-based amplification reaction whereby reagent primers anneal to the target nucleic acid to amplify the target nucleic acid when said target nucleic acid is present in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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Specification