COMPOSITIONS AND KITS FOR MOLECULAR COUNTING
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.
1 .- 163 . (canceled)
164 . A composition for counting the number of occurrences of one or more mRNA target molecules, comprising:
a plurality of first labeled double-stranded DNA molecules corresponding to copies of a first mRNA target molecule, wherein each of the plurality of first labeled double-stranded DNA molecules comprises:
a target specific sequence comprising an oligo dT sequence,
a unique identifier sequence,
a universal PCR primer sequence, and
a copy of the first mRNA target molecule, a complement thereof, a reverse complement thereof, or a portion of any of the preceding,
wherein each of the plurality of first labeled double-stranded DNA molecules is produced by nested PCR using the universal PCR primer sequence
wherein the unique identifier sequence uniquely identifies occurrences of the mRNA target molecule, and
wherein the number of different unique identifier sequences of the plurality of first labeled double-stranded DNA molecules indicates the number of occurrences of the first mRNA target molecule.
165 . The composition of claim 164 , wherein the unique identifier sequence is at least 5 nucleotides in length.
166 . The composition of claim 164 , wherein the target specific sequence is at least 10 nucleotides in length.
167 . The composition of claim 164 , wherein the unique identifier sequence comprises a random sequence.
168 . The composition of claim 164 , wherein the unique identifier sequence comprises a predetermined sequence.
169 . The composition of claim 164 , wherein the number of first labeled double-stranded DNA molecules of the plurality of first labeled double-stranded DNA molecules with different unique identifier sequences is at least 100.
170 . The composition of claim 164 , further comprising a plurality of second labeled double-stranded DNA molecules corresponding to copies of a second mRNA target molecule, wherein each of the plurality of second labeled double-stranded DNA molecules is produced by nested PCR, and comprises:
the target specific sequence comprising the oligo dT sequence,
the unique identifier sequence, and
a copy of one of the second mRNA target molecules, a complement thereof, a reverse complement thereof, or a portion of any of the preceding,
wherein the unique identifier sequences uniquely identify occurrences of the second mRNA target molecules, and
wherein the number of different unique identifier sequences of the plurality of second labeled double-stranded DNA molecules indicates the number of occurrences of the second mRNA target molecule.
171 . The composition of claim 170 , wherein the number of second labeled double-stranded DNA molecules of the plurality of second labeled double-stranded DNA molecules with different unique identifier sequences is at least 1000.
172 . The composition of claim 170 , wherein the first mRNA target molecule and the second mRNA target molecule comprise mRNA target molecules of a single cell.
173 . The composition of claim 171 , wherein the first mRNA target molecule and the second mRNA target molecule comprise mRNA target molecules of a single cell.
174 . The composition of claim 172 , wherein the single cell is a cancer cell, a healthy cell, or an infected cell.
175 . The composition of claim 172 , wherein the single cell is a mammalian cell.
176 . The composition of claim 170 , wherein the number of second labeled double-stranded DNA molecules of the plurality of second labeled double-stranded DNA molecules with different unique identifier sequences is at least 100.
177 . A kit for counting the number of occurrences of one or more mRNA target molecules, comprising:
a plurality of oligonucleotide tags, wherein each oligonucleotide tag of the plurality of oligonucleotide tags comprises:
a target specific sequence comprising an oligo dT sequence,
a unique identifier sequence,
a universal PCR primer sequence, and
wherein at least 1000 of the plurality of oligonucleotide tags comprise different unique identifier sequence;
a universal primer capable of binding to the oligonucleotide tag or the universal primer binding site of the oligonucleotide tag; and
an enzyme.
178 . The kit of claim 177 , wherein the enzyme is a reverse transcriptase, a polymerase, or a combination thereof.
179 . The kit of claim 177 , wherein the plurality of oligonucleotide tags are associated with a solid support.
180 . The kit of claim 179 , wherein the solid support is in the form of beads, resins, gels, microspheres, or a combination thereof.
181 . The kit of claim 179 , wherein the solid support comprises a bead.
182 . The kit of claim 177 , wherein the plurality of oligonucleotide tags are associated with a semisolid support.
183 . The kit of claim 182 , wherein the semisolid support comprises a bead.