Dynamic Array Assay Methods

Abstract

High throughput methods are used that combine the features of using a matrix-type microfluidic device, labeled nucleic acid probes, and homogenous assays to detect and/or quantify nucleic acid analytes. The high throughput methods are capable of detecting nucleic acid analyses with high PCR and probe specificity, producing a low fluorescence background and therefore, a high signal to noise ratio. Additionally, the high throughput methods are capable of detecting low copy number nucleic acid analyte per cell.

What is claimed is: 
     
         1 . An assay method for detection of a plurality of nucleic acid analytes, said method comprising the steps of:
 combining aliquots of a nucleic acid-containing sample with labeled nucleic acid probes and PCR primers in separate compartments of a microfluidic device, wherein said compartments are arrayed in columns and rows, and segregating the aliquots from each other,   wherein said combining comprises
 introducing into each column a column solution, wherein at least two columns do not receive the same column solution, wherein each column solution comprises a unique plurality of reverse primers and one or more probes, 
 introducing into each row a row solution, wherein at least two rows do not receive the same row solution, wherein each row solution comprises a unique plurality of forward primers 
 in each compartment combining a column solution and a row solution 
 wherein the primers in each column solution and the primers in each row solution are selected to provide a plurality of forward-reverse primer combinations, each combination being capable of amplifying a unique region of said nucleic acid to produce a plurality of amplicons, and 
 wherein the probe(s) in each column solution comprise a sequence that specifically bind to one or more of said amplicons, and in aggregate the probe(s) in each column solution bind each amplicon in said plurality, 
   performing a homogeneous assay; and
 querying each of the samples for the presence of a target nucleic acid analyte. 
   
     
     
         2 . The method of  claim 1 , further comprising the step of performing a preliminary amplification reaction on the nucleic acid containing sample to generate a pre-amplified sample. 
     
     
         3 . The method of  claim 2 , wherein the preliminary amplification reaction comprises making a reaction mix containing the nucleic acid containing sample, the forward primers, and the reverse primers and subjecting the reaction mix to PCR amplification. 
     
     
         4 . The method of  claim 3 , wherein the reaction mix is subjected in a range of about 10 cycles to about 18 cycles of PCR amplification. 
     
     
         5 . The method of  claim 4 , wherein the reaction mix is subjected to about 14 cycles of PCR amplification. 
     
     
         6 . The method of  claim 1 , wherein the nucleic acid is cDNA. 
     
     
         7 . The method of  claim 1 , wherein the homogenous assay in said performing step is conducted under conditions which promotes hybridization of the probes to the target nucleic acid analyte. 
     
     
         8 . The method of  claim 1 , wherein the homogeneous assay is performed under conditions which promotes amplification of the target nucleic acid analyte. 
     
     
         9 . The method of  claim 1 , wherein the homogeneous assay comprises performing PCR. 
     
     
         10 . The method of  claim 9 , wherein the PCR is real time PCR. 
     
     
         11 . The method of  claim 1 , wherein the probe in said combining step is a molecular probe capable of detecting about 8-mer or about 9-mer motifs on the target nucleic acid analyte. 
     
     
         12 . The method of  claim 11 , wherein the 8-mer or 9-mer motifs on the target nucleic acid analyte are flanked on one side by the forward primer and on the other side by the reverse primer. 
     
     
         13 . The method of  claim 1 , wherein the probe in said combining step is labeled on a 5′ end with a fluorescent molecule and labeled on a 3′ end with a dark quencher dye. 
     
     
         14 . The method of  claim 1 , wherein the microfluidic device comprises a 48×48 array of reaction chambers. 
     
     
         15 . The method of  claim 1 , wherein the microfluidic device comprises a 96×96 array of reaction chambers. 
     
     
         16 . The method of  claim 1 , wherein at least about 1000 target nucleic acid analytes are detected.