Methods and apparati using single polymer analysis
View Patent ↗The invention relates to methods for analyzing and characterizing single polymers such as nucleic acid molecules. In preferred embodiments, the single molecules are analyzed using single molecule detection and analysis systems.
1. A method for detecting a mutant polymer comprising
contacting a polymer with one or a plurality of unique and distinct mutant-specific unit specific markers each labeled with a first detectable label,
contacting the polymer with a polymer-specific unit specific marker that binds to wild type and mutant polymers and is labeled with a second detectable label, and
analyzing the polymer for coincidence binding of the first and second detectable labels on the polymer by detecting two separate and distinguishable signals from the first and second detectable labels,
wherein the first and second detectable labels are unique and distinct, the polymer is not in vitro amplified, the mutant-specific and polymer-specific unit specific markers are not ligated to each other, and the coincident binding indicates the polymer is a mutant polymer.
2. The method of claim 1 , wherein the polymer is a nucleic acid.
3. The method of claim 2 , wherein the nucleic acid is a DNA or RNA.
4. The method of claim 1 , wherein the mutant-specific unit specific markers are specific for a single nucleotide polymorphism, a deletion, an insertion, a genomic amplification, or an inversion.
5. The method of claim 1 , wherein the first and second detectable labels are of the same type.
6. The method of claim 1 , wherein the first and second detectable labels are fluorescent molecules.
7. The method of claim 1 , wherein the coincident binding is detected by the coincident direct detection of the first and second detectable labels.
8. The method of claim 1 , further comprising a column purification step.
9. The method of claim 1 , wherein the coincident binding yields a color coincident event.
10. The method of claim 1 , wherein the polymer is present in a nanoliter volume.
11. The method of claim 1 , wherein the polymer is present at a frequency of 1 in 1,000,000 molecules in a sample.
12. The method of claim 1 , wherein the unit specific markers are comprised of DNA, RNA, PNA, LNA or a combination thereof.
13. The method of claim 1 , wherein unbound detectable labels are not removed prior to analyzing the polymer for coincidence binding of the first and second detectable labels on the polymer.
14. The method of claim 1 , wherein the first and second detectable labels are provided as molecular beacon probes.
15. The method of claim 1 , wherein at least the first or the second detectable label is attached to a nucleic acid molecule hybridized to a universal linker attached to a unit specific marker.
16. The method of claim 1 , wherein the polymer is fixed to a solid support in a random orientation.
17. The method of claim 1 , wherein the polymer is fixed to a solid support in a non-continuous manner.
18. The method of claim 1 , wherein the binding of the mutant-specific unit specific marker and the polymer-specific unit specific marker to the polymer is determined by confocal detection.
19. The method of claim 1 , wherein detection of coincident binding of both the polymer-specific unit specific marker and any one or more of the mutant-specific unit specific markers indicates the polymer is a mutant polymer.
20. The method of claim 1 , wherein the polymer is a single polymer.
21. A method for detecting a mutant polymer comprising
contacting a polymer-containing sample with one or a plurality of unique and distinct mutant-specific unit specific markers each labeled with a first detectable label,
contacting the sample with a polymer-specific unit specific marker that binds to wild type and mutant polymers and is labeled with a second detectable label, wherein the first and second detectable labels are unique and distinct, and
detecting a single complex of a polymer and first and second detectable labels as an indicator of a single mutant polymer, by detecting two separate and distinguishable signals from the first and second detectable labels.
22. A method for detecting a mutant polymer comprising
contacting a polymer with one or a plurality of unique and distinct mutant-specific unit specific markers each labeled with a first detectable label,
contacting the polymer with a polymer-specific unit specific marker that binds to wild type and mutant polymers and is labeled with a second detectable label, and
analyzing the polymer for coincidence binding of the first and second detectable labels on the polymer,
wherein the first and second detectable labels are unique and distinct, the polymer is not in vitro amplified, and the coincident binding indicates the polymer is a mutant polymer, and the coincident binding yields a color coincident event.
23. A method for detecting a mutant polymer comprising
contacting a polymer with one or a plurality of unique and distinct mutant-specific unit specific markers each labeled with a first detectable label,
contacting the polymer with a polymer-specific unit specific marker that binds to wild type and mutant polymers and is labeled with a second detectable label, and
analyzing the polymer for coincidence binding of the first and second detectable labels on the polymer,
wherein the first and second detectable labels are unique and distinct, the polymer is not in vitro amplified, and the coincident binding indicates the polymer is a mutant polymer, and at least the first or the second detectable label is attached to a nucleic acid molecule hybridized to a universal linker attached to a unit specific marker.