Methods of detecting low copy nucleic acids
Inventors
Knapp, Michael R. • Baker, Jill M. • Chow, Andrea W. • Kopf-Sill, Anne R. • Spaid, Michael A.
Assignees
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Publication Number
US-8697362-B2
Publication Date
2014-04-15
Expiration Date
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.
Core Innovation
The invention provides a method of detecting a nucleic acid of interest by aliquotting a sample comprising the nucleic acid into a plurality of reaction mixtures, where at least one reaction mixture is a single copy reaction mixture comprising a single copy of the nucleic acid of interest. The reaction mixtures are flowed throughout a network of microchannels, and then subjected to one or more amplification reactions under stopped flow conditions in an amplification area of the network of microchannels.
Detection is performed in the at least one single copy reaction mixture under the stopped flow conditions in the amplification area in which the single copy reaction mixture was subjected to the one or more amplification reactions. The approach is implemented in microfluidic architectures that include a network of microchannels and microscale amplification chambers or channels.
In additional embodiments, the amplification reactions include one or more non-thermal amplification reactions in one or more microscale amplification chamber or channel. Quantification and interpretation of low-copy nucleic acids are supported by reproducible signal peak shapes/dispersion and probabilistic/statistical interpretation, where the presence of positive aliquots is used together with probabilistic modeling and other computational/analytical techniques for low-copy and single-molecule detection.
Claims Coverage
The independent claims identified are clm-00001 and clm-00035, covering methods that aliquot a sample into single-copy reaction mixtures and detect after stopped-flow amplification in a microchannel network, and perform non-thermal amplification in microscale amplification chambers or channels. The inventive features below reflect the main claim elements as stated.
Aliquoting into single copy reaction mixtures and microchannel network flow
Aliquotting a sample comprising the nucleic acid of interest into a plurality of reaction mixtures, wherein at least one of the reaction mixtures is a single copy reaction mixture comprising a single copy of the nucleic acid of interest; flowing the reaction mixtures throughout a network of microchannels.
Stopped flow amplification and detection in the same microchannel amplification area
Subjecting the plurality of reaction mixtures to one or more amplification reactions under stopped flow conditions in an amplification area of the network of microchannels; and detecting the nucleic acid of interest in the at least one single copy reaction mixture under said stopped flow conditions in the amplification area of the network of microchannels in which the at least one single copy reaction mixture was subjected to one or more amplification reactions, wherein the plurality of reaction mixtures are not further flowed between the subjecting and the detecting.
Non-thermal amplification in microscale amplification chambers or channels
Subjecting the reaction mixtures to one or more amplification reactions comprises performing one or more non-thermal amplification reactions in one or more microscale amplification chamber or channel.
Overall, the claims cover microfluidic detection of nucleic acids at single-copy level by aliquotting into single-copy reaction mixtures, flowing through a microchannel network, performing amplification under stopped-flow conditions in a designated microchannel amplification area, and detecting without further flowing; with additional coverage for non-thermal amplification implemented in microscale amplification chambers or channels.
Stated Advantages
Documented Applications
SNP allele typing using the described nucleic acid detection and quantification approach.
Cancer marker detection, including detection of KRAS and p53.
Virus detection with stop-flow imaging and counting of single-copy amplicons.
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