Analysis using microfluidic partitioning devices

Abstract

The invention relates to methods, reagents and devices for detection and characterization of nucleic acids, cells, and other biological samples. Assay method are provided in which a sample is partitioned into sub-samples, and analysis of the contents of the sub-samples carried out. The invention also provides microfluidic devices for conducting the assay. The invention also provides an analysis method using a universal primers and probes for amplification and detection.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims benefit of U.S. provisional application No. 60 / 687,010, filed Jun. 2, 2005, the entire contents of which are herein incorporated by reference.FIELD OF THE INVENTION[0002]The invention relates to methods, reagents and devices for detecting and characterizing nucleic acids, cells, and other biological samples.BACKGROUND[0003]A variety of nucleic acid amplification assays and immunological assays are used for analysis of cells and nucleic acids. These assays can be used to detect or characterize nucleic acid sequences associated with particular diseases or genetic disorders, for genotyping, for gene expression analyses, to detect and identify pathogens such as viruses, bacteria and fungi), for paternity and forensic identification, and for many other purposes. However, in some applications the efficiency and sensitivity of these assays is reduced, which may render the assays useless or at minimum require that addition...

AI Technical Summary

Benefits of technology

[0005]In a related aspect, the invention provides an assay method including the following steps (a) partitioning a sample into a plurality of sub-samples, where said sample comprises a plurality of nucleic acid molecules, and where at least two sub-samples comprise at least one nucleic acid molecule; (b) providing sufficient reagents in each sub-sample to amplify at least two different target sequences; (c) amplifying target sequence(s) in at least two sub-sample(s) thereby producing amplicons in the sub-sample(s); (d) combining the amplicons from said at least two sub-samples to create an amplicon pool; (d) dividing the amplicon pool into a plurality of aliquots; and, (e) for each aliquot, determining a property of amplicons in the aliquot. In one embodiment, the sample is partitioned into at least 104 sub-samples. In one embodiment, each subsample has a volume of less than one nanoliter. In one embodiment, the nucleic acid molecules comprise DNA and/or mRNA. In one embodiment, the amplification is by PCR or RT-PCR. In one embodiment, sufficient reagents are provided to amplify at least 10, 20, or 50 different target sequences, if present. In one embodiment, the amplicon pool is divided into at least 10, 20, 50 or 100 aliquiots. In one embodiment, the sample contains a plurality of cells having nuclei...

Problems solved by technology

However, in some applications the efficiency and sensitivity of these assays is reduced, which may render the assays useless or at minimum require that additional manipulations and / or significant amounts of expensive reagents be used.

For example, when a cell or molecule to be analyzed is from a sample with a large excess of non-target cells or molecules (e.g., as in genetic or phenotypic analysis of a rare cell in a background of other cells) conventional assay methods are inadequate.

Similarly, when a number of different targets must be detected in a single sample, conventional approaches (e.g., multiplex PCR) are expensive, inefficient or not sufficiently sensitive.

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