Compositions and kits for molecular counting
AssigneeBecton, Dickinson and Company
InventorsGlenn K. Fu, Stephen P. A. Fodor, Julie Wilhelmy
Life of this patent
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Abstract
Methods, kits and systems are disclosed for analyzing one or more molecules in a sample. Analyzing the one or more molecules may comprise quantitation of the one or more molecules. Individual molecules may quantitated by PCR, arrays, beads, emulsions, droplets, or sequencing. Quantitation of individual molecules may further comprise stochastic labeling of the one or more molecules with a plurality of oligonucleotide tags to produce one or more stochastically labeled molecules. The methods may further comprise amplifying, sequencing, detecting, and/or quantifying the stochastically labeled molecules. The molecules may be DNA, RNA and/or proteins.
Claims20
A method for the absolute quantification of copies of mRNA molecules in a sample, comprising:
- (a) stochastically labeling mRNA copies of a gene of interest in a sample with a plurality of oligonucleotide tags to produce a plurality of labelled-mRNA copies, wherein:
- (i) the number of mRNA copies of the gene of interest is at least one;
- (ii) the plurality of oligonucleotide tags each comprises an oligodT sequence, a universal primer binding site, and an identifier region;
- (iii) the plurality of oligonucleotide tags comprises at least 100 oligonucleotide tags having identifier regions of different sequences for determining the number of mRNA copies of the gene of interest;
- and
- (iv) a number of oligonucleotide tags having identifier regions of different sequences in the plurality of oligonucleotide tags is at least 5 times greater than the number of mRNA copies of any of the two or more genes of interest;
- (b) conducting a first strand synthesis reaction by contacting the plurality of labelled-mRNA copies with a reverse transcriptase enzyme to produce a plurality of single-stranded labelled-cDNA molecules;
- (c) amplifying the plurality of single-stranded labelled-cDNA molecules to produce a plurality of double-stranded labelled-cDNA molecules;
- (d) conducting a nested PCR reaction on the plurality of double-stranded labelled-cDNA molecules to produce a plurality of nested PCR labelled-amplicons;
- and
- (e) detecting at least a portion of amplicons of the nested PCR labelled-amplicons to count the number of different identifier regions associated with the labeled mRNA copies, thereby counting the absolute number of mRNA copies of the gene of interest in the sample.
Dependencies are inferred from the claim text.