METHODS OF DETECTING LOW COPY NUCLEIC ACIDS
First Claim
1. A method of detecting a nucleic acid of interest, the method comprising:
- aliquotting a sample comprising the nucleic acid of interest and one or more additional nucleic acids into a plurality of reaction mixtures, wherein at least two of the reaction mixtures are single copy reaction mixtures, each comprising a single copy of the nucleic acid of interest, wherein the plurality of reaction mixtures additionally comprise at least one additional reaction mixture comprising at least one copy of the additional nucleic acid;
subjecting the plurality of reaction mixtures to one or more amplification reactions while flowing the reaction mixtures through a channel of a microfluidic device; and
, detecting the nucleic acid of interest in the single copy reaction mixtures.
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Accused Products
Abstract
Methods are provided for detecting low copy nucleic acids of interest in a sample. In one method, a sample comprising a nucleic acid of interest is aliquotted into a plurality of reaction mixtures, at least two of which are single-copy reaction mixtures. The reaction mixtures are subjected to one or more amplification reactions while flowing through a channel of a microfluidic device. At least one of the reaction mixtures is formulated in an aqueous phase of an emulsion comprising aqueous droplets suspended in an immiscible liquid. The nucleic acid of interest is present as a single copy in at least one aqueous droplet of the aqueous phase prior to performing the amplification reaction(s). Amplification is performed on the reaction mixture when it is formulated in the emulsion. The nucleic acid is continuously flowed during a plurality of steps of the method.
185 Citations
25 Claims
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1. A method of detecting a nucleic acid of interest, the method comprising:
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aliquotting a sample comprising the nucleic acid of interest and one or more additional nucleic acids into a plurality of reaction mixtures, wherein at least two of the reaction mixtures are single copy reaction mixtures, each comprising a single copy of the nucleic acid of interest, wherein the plurality of reaction mixtures additionally comprise at least one additional reaction mixture comprising at least one copy of the additional nucleic acid;
subjecting the plurality of reaction mixtures to one or more amplification reactions while flowing the reaction mixtures through a channel of a microfluidic device; and
,detecting the nucleic acid of interest in the single copy reaction mixtures. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method of detecting a low copy nucleic acid of interest in a sample that comprises a higher copy additional nucleic acid that is different from the low copy nucleic acid, the method comprising:
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aliquotting the sample into a plurality of reaction mixtures, which reaction mixtures comprise a plurality of zero copy reaction mixtures comprising zero copies of the nucleic acid of interest and at least one single copy reaction mixture comprising a single copy of the nucleic acid of interest;
subjecting the zero and single copy reaction mixtures to an amplification reaction while flowing the reaction mixtures through a channel of a microfluidic device;
detecting the nucleic acid of interest in the single copy reaction mixture. - View Dependent Claims (10, 11, 12, 13, 14, 15)
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16. A method of quantifying a nucleic acid of interest in a sample, the method comprising:
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aliquotting a sample comprising a copy of the nucleic acid of interest, or a complement thereof, into at least one reaction mixture;
subjecting the reaction mixture to at least one amplification reaction, thereby amplifying the copy of the nucleic acid of interest;
detecting a shape, volume, width, length, height, or area in which the nucleic acid of interest, or a signal corresponding thereto, is present; and
,correlating the shape, volume, width, height, length, or area to a number of copies of the nucleic acid of interest in the reaction mixture or sample, thereby quantifying the nucleic acid of interest in the sample. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23)
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24. A method of detecting a nucleic acid of interest, the method comprising:
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aliquotting a sample comprising the nucleic acid of interest into a plurality of reaction mixtures, wherein at least two of the reaction mixtures are single copy reaction mixtures, each comprising a single copy of the nucleic acid of interest;
flowing the reaction mixtures throughout a network of microchannels;
subjecting the plurality of reaction mixtures to one or more amplification reactions under stopped flow conditions in the network of microchannels; and
,detecting the nucleic acid of interest in the single copy reaction mixtures under said stopped flow conditions. - View Dependent Claims (25)
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Specification