Methods of detecting target nucleic acids
The present disclosure relates to methods of identifying target nucleic acids by using coded molecules and its analysis by translocation through a nanopore. Generally, coded molecules are subject to a target polynucleotide dependent modification. The modified coded molecule is detected by isolating the modified coded molecules from the unmodified coded molecules prior to analysis through the nanopore or by detecting a change in the signal pattern of the coded molecule when analyzed through the nanopore.
1. A method of detecting a target polynucleotide, comprising:
a) contacting a coded molecule with a target polynucleotide, wherein the coded molecule comprises
(i) one or more block polymer regions, and
(ii) a target probe capable of hybridizing to the target polynucleotide;
b) modifying the coded molecule by modifying the target probe with a modifying agent following hybridizing of the target probe with the target polynucleotide, wherein the modified target probe is indicative of the target polynucleotide hybridized to the target probe;
c) translocating the modified coded molecule through a nanopore and detecting a signal pattern associated with the modified coded molecule; and
d) comparing the detected signal pattern to a signal pattern of an unmodified coded molecule, wherein a difference in the detected signal pattern of the modified coded molecule compared to the signal pattern of the unmodified coded molecule indicates the presence of the target polynucleotide.
2. The method of claim 1 , further comprising associating the detected signal pattern to the target probe.
3. The method of claim 1 , in which the target polynucleotide comprises a 5-prime region and a 3-prime region, and the target probe comprises a 3-prime terminal sequence that hybridizes to the 5-prime region of the target polynucleotide, and wherein the modifying agent is a template-dependent polymerase and the modification is extension of the hybridized 3-prime region of the target probe.
4. The method of claim 3 , in which the 3-prime terminal sequence of the target probe comprises a 3-prime terminal nucleotide that interrogates a site of nucleotide polymorphism on the target polynucleotide.
5. The method of claim 3 , in which the target polynucleotide comprises a circular nucleic acid, wherein the circular nucleic acid is a ligated open circle probe (OCP).
6. The method of claim 1 , in which the target polynucleotide comprises adjacent first and second regions, and the method further comprises hybridizing a ligation probe to the target polynucleotide, wherein the ligation probe hybridizes to the first region and the target probe hybridizes to the second region of the target polynucleotide such that a terminus of the ligation probe and a terminus of the target probe are adjacent, and wherein the modifying agent is a ligase and the modification is ligation of the ligation probe to the target probe.
7. The method of claim 6 , in which the terminus of the target probe comprises a terminal nucleotide that interrogates a site of nucleotide polymorphism on the target polynucleotide.
8. The method of claim 6 , in which the terminus of the ligation probe comprises a terminal nucleotide that interrogates a site of nucleotide polymorphism on the target polynucleotide.
9. The method of claim 1 , in which the target polynucleotide comprises adjacent first and second regions, and the target probe comprises a 5-prime region and a 3-prime region, and wherein the method further comprises hybridizing a FLAP probe to the target polynucleotide, wherein the FLAP probe comprises a 3-prime segment that hybridizes to the first region, and the 3-prime region of the target probe hybridizes to the second region such that the 3-prime segment of the FLAP probe and the 3-prime region of the target probe are adjacently hybridized to the target polynucleotide to form a FLAP substrate, and wherein the modifying agent is a FLAP endonuclease and the modification is cleavage of the target probe.
10. The method of claim 9 , in which the 5-prime region of the target probe is non-complementary to the target polynucleotide.
11. The method of claim 9 , in which the FLAP probe further comprises a 3-prime unpaired segment that overlaps with the 5-prime region of the target probe in the FLAP substrate, thereby forming a double FLAP substrate.
12. The method of claim 9 , in which the target probe interrogates a site of nucleotide polymorphism on the target polynucleotide.
13. The method of claim 9 , in which the FLAP probe interrogates a site of nucleotide polymorphism on the target polynucleotide.
14. The method of claim 1 , in which the hybridization of the target probe to the target polynucleotide forms an endonuclease recognition site and a corresponding enodonuclease cleavage site, and wherein the modifying agent is an endonuclease that recognizes the recognition site and the modification is cleavage of the target probe.
15. The method of claim 14 , in which the endonuclease recognition site is a sequence-specific endonuclease recognition site and wherein the modifying agent is a sequence specific endonuclease active on the recognition site.
16. The method of claim 1 , in which the modifying agent is a double-stranded specific exonuclease suitable to act on the target probe hybridized to the target polynucleotide and the modification is degradation of all or a portion of the target probe.
17. The method of claim 1 , in which the coded molecule comprises a chimeric polymer.
18. The method of claim 17 , in which the chimeric polymer comprises nucleobase and non-nucleobase polymers.
19. The method of claim 1 , in which the coded molecule comprises at least two block polymers, and wherein the two block polymers are separated by a non-block polymer segment.
20. The method of claim 1 , in which the block polymer comprises dinucleotide repeats.