Methods for detection and quantification of nucleic acid or protein targets in a sample
View Patent ↗The invention provides an assay method for detection and/or quantification of a plurality of nucleic acid or protein targets in a sample. In the method probes are used to associate a detectable tag sequence with each of the selected targets present in the sample. Probes or primers sufficient to identify at least 25, and preferably at least 500, different targets are used. The method involves segregating aliquots of the sample from each other and detecting the tag sequences in each aliquot.
1. An assay method for detection of a plurality of nucleic acid or protein targets in a sample comprising
(a) combining target-specific probes with the sample under conditions in which at least 25 target-specific probes each bind a different specific target in the sample, if the specific target is present,
wherein each said target-specific probe comprises a common nucleic acid tag sequence and a unique combination of a first primer binding site and a second primer binding site flanking the nucleic acid tag sequence,
wherein at least two of said target-specific probes do not have the same first primer binding sites and at least two of said target-specific probes do not have the same second primer binding sites, and
wherein the target-specific probe forms a target-specific probe product associated with the target if the target is present in the sample, thereby associating the tag with the target by forming a target-specific probe product associated with the target;
(b) distributing aliquots of the sample to separate compartments of a microfluidic device and segregating the aliquots from each other;
(c) querying each said aliquot for the presence or absence of a different target-specific probe product, wherein said querying comprises amplifying the nucleic acid tag sequence using a first primer that binds the first primer binding site and a second primer that binds the second primer binding site; and detecting the presence or absence of the common nucleic acid tag sequence using a tag-detecting probe.
2. The method of claim 1 wherein target-specific probes sufficient to identify at least 500 different targets are combined with the sample.
3. The method of claim 1 wherein said querying comprises quantifying the amount of the nucleic acid tag sequence in each aliquot and correlating the amount of the tag sequence with the amount of target in the sample.
4. The method of claim 3 in which the tag sequence is amplified using PCR.
5. The method of claim 4 wherein the amplified tag sequence is detected using a fluorogenic nuclease assay.
6. The method of claim 5 wherein the amplified tag sequence is quantified by the continuous measurement of PCR product accumulation using a dual-labeled fluorogenic oligonucleotide probe.
7. The method of claim 1 wherein the target is a nucleic acid molecule.
8. The method of claim 7 wherein the target-specific probe is a padlock probe.
9. The method of claim 1 wherein the target is a protein molecule.
10. The method of claim 1 wherein the target-specific probe is a proximity probe.
11. The method of claim 1 wherein the assay has a dynamic range of at least 4 orders of magnitude.
12. The method of claim 1 wherein the microfluidic device is fabricated, at least in part, from an elastomeric material.
13. The method of claim 1 wherein querying each aliquot of the sample for the presence of a different target-specific probe comprises combining each aliquot with a composition containing PCR primers and/or a tag detecting probe.
14. The method of claim 13 wherein querying each aliquot of the sample for the presence of a different target-specific probe comprises combining each aliquot with a composition containing PCR primers and a tag detecting probe.
15. The method of claim 14 wherein the tag-detecting probe is a dual-labeled fluorogenic oligonucleotide probe.
16. The method of claim 15 wherein each aliquot is queried using the same tag-detecting probe and a different PCR primer pair.
17. The method of claim 15 wherein each aliquot is queried using the same PCR primer pair and a different tag-detecting probe.
18. An assay method for detection of a plurality of DNA targets in a sample comprising
combining target-specific padlock probes sufficient to identify at least 25 different targets with the sample under conditions in which the probes bind and cantenate to the target molecules, if present, forming a target-specific probe product
wherein each probe comprises a common nucleic acid tag sequence and a unique combination of a first primer binding site and a second primer binding site wherein at least two of said target-specific probes do not have the same first primer binding sites and at least two of said target-specific probes do not have the same second primer binding sites, and wherein the padlock probes form target-specific probe products associated with the target if the target is present in the sample, thereby associating the tag sequence with the target
distributing aliquots of the sample to separate compartments of a microfluidic device and segregating the aliquots from each other;
querying each aliquot for the presence of a different probe molecule catenated to the target, wherein said querying comprises detecting the tag sequence if present in the aliquot, and wherein detecting the tag comprises amplifying the tag sequence using a first primer that binds the first primer binding site and a second primer that binds the second primer binding site and detecting the amplification using a dual-labeled fluorogenic oligonucleotide probe.