Multiplex branched-chain DNA assays
Methods of detecting two or more nucleic acids in a multiplex branched-chain DNA assay are provided. Different nucleic acids are captured through cooperative hybridization events on different, identifiable subsets of particles or at different selected positions on a spatially addressable solid support. Compositions, kits, and systems related to the methods are also described.
1. A composition for detecting ten or more nucleic acids of interest, the composition comprising:
a single mixture, wherein the single mixture comprises:
ten or more subsets of particles, a plurality of the particles in each subset being distinguishable from a plurality of the particles in every other subset, and the particles in each subset having associated therewith a different capture probe;
ten or more subsets of n capture extenders, wherein n is at least two, wherein each subset of n capture extenders is capable of hybridizing to one of the nucleic acids of interest, and wherein the capture extenders in each subset are capable of hybridizing to one of the capture probes and thereby associating each subset of n capture extenders with a selected subset of the particles;
wherein the capture probes and the capture extenders comprise only naturally occurring bases A, C, G, T, and/or U;
ten or more subsets of one or more label extenders; and
a label probe system comprising a label.
2. The composition of claim 1 , comprising one or more of the nucleic acids of interest, wherein each nucleic acid of interest is hybridized to its corresponding subset of n capture extenders, and the corresponding subset of n capture extenders is hybridized to its corresponding capture probe; and wherein the composition is maintained at a hybridization temperature which is greater than a melting temperature T m of a complex between each individual capture extender and the corresponding capture probe.
3. The composition of claim 2 , wherein at least one of the nucleic acids of interest is present in the composition in a non-zero amount of 200 amol or less, 150 amol or less, 100 amol or less, 50 amol or less, 10 amol or less, 1 amol or less, or 0.1 amol or less.
4. The composition of claim 1 , wherein the composition comprises a first nucleic acid of interest and a second nucleic acid, the second nucleic acid having a polynucleotide sequence which is 95% or more identical to that of the first nucleic acid, wherein the first nucleic acid is captured on a first subset of particles, and wherein the second nucleic acid comprises 1% or less of a total amount of nucleic acid captured on the first subset of particles.
5. The composition of claim 1 , wherein the ten or more nucleic acids of interest comprise 20 or more nucleic acids of interest, wherein the ten or more subsets of particles comprise 20 or more subsets of particles, wherein the ten or more subsets of n capture extenders comprise 20 or more subsets of n capture extenders, and wherein the ten or more subsets of label extenders comprise 20 or more subsets of label extenders.
6. The composition of claim 1 , wherein the ten or more nucleic acids of interest comprise 30 or more nucleic acids of interest; wherein the ten or more subsets of particles comprise 30 or more subsets of particles; wherein the ten or more subsets of n capture extenders comprise 30 or more subsets of n capture extenders; and wherein the ten or more subsets of label extenders comprise 30 or more subsets of label extenders.
7. The composition of claim 1 , wherein the particles are microspheres.
8. The composition of claim 7 , wherein the microspheres of each subset are distinguishable from those of the other subsets on the basis of their fluorescent emission spectrum, their diameter, or a combination thereof.
9. The composition of claim 1 , wherein n is at least three.
10. The composition of claim 1 , wherein the n capture extenders in a subset hybridize to nonoverlapping polynucleotide sequences in the corresponding nucleic acid of interest.
11. The composition of claim 1 , wherein each capture extender comprises a polynucleotide sequence C-1 that is complementary to a polynucleotide sequence C-2 in its corresponding capture probe, and wherein C-1 and C-2 are 20 nucleotides or less in length.
12. The composition of claim 2 , wherein the hybridization temperature is about 5° C. or more greater than the T m .
13. The composition of claim 1 , wherein the label probe system comprises an amplification multimer and a plurality of label probes, wherein the amplification multimer is capable of hybridizing to a label extender and to a plurality of label probes.
14. The composition of claim 13 , wherein the label probe comprises the label.
15. The composition of claim 1 , comprising the ten or more nucleic acids of interest, which ten or more nucleic acids of interest comprise ten or more mRNAs.
16. The composition of claim 1 , wherein the composition comprises a first nucleic acid of interest and a second nucleic acid, the first nucleic acid being a first splice variant and the second nucleic acid being a second splice variant, wherein a first subset of n capture extenders is hybridized to the first splice variant, whereby the first splice variant is captured on a first subset of particles, and wherein at most n−1 of the capture extenders are hybridized to the second splice variant, whereby the second splice variant is not captured on the first subset of particles.