Assay methods for increased throughput of samples and/or targets

Abstract

The present invention provides assay methods that increase the number of samples and/or target nucleic acids that can be analyzed in a single assay.

1 - 12 . (canceled) 
     
     
         13 . An assay method for detecting a plurality of target nucleic acids in a sample, the method comprising:
 providing T forward preamplification primers to a sample, wherein each forward preamplification primer comprises a different target-specific nucleotide sequence and a set-specific nucleotide tag, wherein X different forward set-specific nucleotide tags are employed, wherein T is the number of targets to be detected, and X is an integer that is greater than 1 and less than T, whereby T/X primers comprise the same forward set-specific nucleotide tag;   providing T reverse preamplification primers to a sample, wherein each reverse preamplification primer comprises a different target-specific nucleotide sequence and a reverse set-specific nucleotide tag, wherein Y different reverse set-specific nucleotide tags are employed, and Y is an integer that is greater than 1 and less than T, whereby T/Y primers comprise the same reverse set-specific nucleotide tag;   subjecting the sample to preamplification to produce an assay mixture, wherein any preamplification product produced for a particular target incorporates a unique combination of forward and reverse set-specific nucleotide tags;   subjecting the assay mixture, or aliquots thereof, to amplification using amplification primers wherein each amplification primer pair comprises:
 a forward amplification primer that anneals to the forward set-specific nucleotide tag; and 
 a reverse amplification primer that anneals to the reverse set-specific nucleotide tag; and 
   for each unique primer pair, determining whether an amplification product is present in the amplification mixture, or aliquot thereof, whereby the presence of an amplification product indicates the presence of a particular target nucleic acid in a particular sample.   
     
     
         14 . The method of  claim 13 , wherein said amplification comprises dividing the assay mixture into T amplification mixtures, and separately amplifying each of said amplification mixtures using a unique pair of amplification primers. 
     
     
         15 . The assay method of  claim 13 , wherein X is at least a value selected from 12, 24, 48, and 96. 
     
     
         16 . The assay method of  claim 13 , wherein the T is at least a value selected from the group consisting of 384, 576, 768, 1152, 2304, 3600, 4608, and 9216. 
     
     
         17 . An assay method for detecting a plurality of target nucleic acids in a sample, the method comprising:
 dividing a sample into R aliquots, wherein R is an integer greater than 1;   separately subjecting each of said R aliquots to an encoding reaction that produces a set of T tagged target nucleotide sequences, wherein T is the number of target nucleic acids to be detected in each aliquot, T being an integer greater than 1, and wherein:
 each tagged target nucleotide sequence comprises a first nucleotide tag 5′ of a target nucleotide sequence, a target nucleotide sequence, and a second nucleotide tag 3′ of the target nucleotide sequence; 
 the combination of nucleotide tags in each of said T tagged target nucleotide sequences is unique for every tagged target nucleotide sequence in each aliquot; and 
 the same set of first and second nucleotide tag combinations is used in the encoding reaction in each of the aliquots; 
   separately subjecting each aliquot to amplification using the same set of T different amplification primer pairs for each aliquot, each primer pair comprising a first primer that anneals to the first nucleotide tag and a second primer that anneals to the second nucleotide tag in each tagged target nucleotide sequence; and   for each unique primer pair in each aliquot, determining whether an amplification product is present in the aliquot, whereby the presence of an amplification product indicates the presence of a particular target nucleic acid in the sample.   
     
     
         18 . The assay method of  claim 17 , wherein, prior to amplification:
 each aliquot is divided into T sub-aliquots;   one of said T different amplification primer pairs is combined with each sub-aliquot; and   the sub-aliquots are subjected to separate amplification reactions.   
     
     
         19 . The assay method of  claim 17 , wherein said encoding reaction comprises a preamplification reaction. 
     
     
         20 . The assay method of  claim 19 , wherein the sample is subjected to a pre-preamplification reaction, wherein each of T×R target nucleic acids is preamplified. 
     
     
         21 . The assay method of  claim 20 , wherein primers employed for preamplification are nested relative to primers employed for pre-preamplification. 
     
     
         22 . The assay method of  claim 17 , wherein R and/or T is/are at least a value selected from 12, 24, 48, and 96. 
     
     
         23 . The assay method of  claim 13 , wherein amplification mixtures are formed in or, distributed into, separate compartments of a microfluidic device prior to amplification. 
     
     
         24 . The assay method of  claim 23 , wherein the microfluidic device is fabricated, at least in part, from an elastomeric material. 
     
     
         25 . The assay method of  claim 23 , wherein the assay has a dynamic range of at least 4 orders of magnitude. 
     
     
         26 . The assay method of  claim 13 , wherein the preamplification and/or the amplification is carried out by polymerase chain reaction (PCR). 
     
     
         27 . The assay method of  claim 13 , wherein the preamplification is carried out for 2-20 cycles to introduce the nucleotide tags. 
     
     
         28 . The assay method of  claim 13 , wherein the preamplification is carried out for a sufficient number of cycles to normalize amplicon copy number across targets and across samples. 
     
     
         29 . The assay method of  claim 13 , wherein the presence of an amplification product is determined by quantitative real-time polymerase chain reaction (qPCR). 
     
     
         30 . The assay method of  claim 13 , wherein a universal qPCR probe is employed in the amplification mixtures to detect amplification products. 
     
     
         31 . The assay method of  claim 13 , wherein one or more target-specific qPCR probes is employed in the amplification mixtures to detect amplification products. 
     
     
         32 . The assay method of  claim 13 , wherein one or more tag-specific qPCR probes is employed in the amplification mixtures to detect amplification products. 
     
     
         33 - 34 . (canceled) 
     
     
         35 . The assay method of  claim 13 , additionally comprising quantifying the amount of amplification product in the amplification mixtures. 
     
     
         36 . The assay method of  claim 35 , additionally comprising determining the amount of each target nucleic acid present in each sample. 
     
     
         37 . The assay method of  claim 13 , wherein the assay is performed to determine the copy numbers of the target nucleic acids. 
     
     
         38 . The assay method of  claim 13 , wherein the assay is performed to determine genotypes at loci corresponding to the target nucleic acids. 
     
     
         39 . The assay method of  claim 13 , wherein the assay is performed to determine the expression levels of the target nucleic acids. 
     
     
         40 . The assay method of  claim 13 , additionally comprising, reducing the concentration of preamplification primers prior to carrying out said amplification. 
     
     
         41 . The method of  claim 13 , wherein the sample comprises a genomic DNA sample. 
     
     
         42 . The method of  claim 41 , wherein the preamplification is conducted in the presence of an amount of a blocking agent that is sufficient to increase specific amplification of the target nucleic acid. 
     
     
         43 . The method of  claim 42 , wherein the blocking agent comprises a nucleic acid blocking agent that hybridizes to repetitive sequences in the genomic DNA sample. 
     
     
         44 . The method of  claim 42 , wherein the blocking agent is selected from the group consisting of tRNA, degenerate oligonucleotide primers, repetitive DNA, bovine serum albumin (BSA), and glycogen. 
     
     
         45 . The method of  claim 42 , wherein the blocking agent is present at a concentration in the range of about 0.1 μg/μl to about 40 μg/μl. 
     
     
         46 . The method of  claim 45 , wherein the blocking agent comprises tRNA at a concentration in the range of about 1 μg/μl to about 5 μg/μl.