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.

1 . An assay method comprising:
 (a) partitioning a sample obtained from the blood of a pregnant woman into a plurality of sub-samples,
 wherein said sample comprises a plurality of nucleic acid molecules, and wherein most of said sub-samples comprise either (i) 0 or 1 target nucleic acid sequences or (ii) 0 or 1 cells; 
   (b) providing sufficient reagents in each sub-sample to amplify at least two different target sequences;   (c) amplifying target sequence(s) in the two sub-sample(s)
 thereby producing amplicon(s) in the sub-sample(s); 
   (d) querying the sub-samples for the presence of an amplicon, or a property of an amplicon, to obtain an indication of fetal disease or propensity to disease.   
   
   
       2 - 17 . (canceled) 
   
   
       18 . The method of  claim 1 , wherein nucleic acid molecules in the sample are free in solution. 
   
   
       19 . The method of  claim 1 , wherein the sample comprises fetal cells. 
   
   
       20 . The method of  claim 1 , wherein the indication of fetal disease or propensity to disease comprises a genetic defect selected from the group consisting of a substitution, an amplification, a deletion, and a translocation. 
   
   
       21 . The method of  claim 1 , wherein the sub-samples are queried for a polymorphism. 
   
   
       22 . The method of  claim 21 , wherein the polymorphism comprises a single nucleotide polymorphism (SNP). 
   
   
       23 . The method of  claim 1 , wherein the sub-samples are queried to quantify a target sequence. 
   
   
       24 . The method of  claim 1 , wherein the sub-samples are queried with a fetal-specific probe. 
   
   
       25 . The method of  claim 1 , wherein the sample is partitioned into at least 10 4  sub-samples. 
   
   
       26 . The method of  claim 25 , wherein each sub-sample has a volume of less than one nanoliter. 
   
   
       27 . The method of  claim 1 , wherein said nucleic acid molecules comprise DNA. 
   
   
       28 . The method of  claim 27 , wherein said DNA comprises cDNA made from RNA. 
   
   
       29 . The method of  claim 1 , wherein sufficient reagents are provide to amplify at least 10 target sequences, if present. 
   
   
       30 . The method of  claim 1 , wherein said amplification is by PCR or RT-PCR. 
   
   
       31 . The method of  claim 1 , wherein the sample comprises a plurality of cells comprising nucleic acid molecules, and wherein partitioning the sample comprises partitioning intact cells into a plurality of sub-samples. 
   
   
       32 . The method of  claim 1 , wherein at least 99% of the sub-samples comprise either (i) 0 or 1 target nucleic acid sequences or (ii) 0 or 1 cells.