Methods and kits for nucleic acid sequencing
Various embodiments of the present disclosure generally relate to molecular biological protocols, equipment and reagents for the sequencing of target nucleic acid (DNA, RNA, cDNA, etc) molecules.
1. A method of sequencing a target polynucleotide, comprising:
immobilizing a primer along a nanowire, wherein the nanowire is coupled to a detector configured to detect a change in electrical resistance of the nanowire;
hybridizing the target polynucleotide to the primer to form a primer-target polynucleotide immobilized along the nanowire;
adding a nucleotide and a polymerase to the primer-target polynucleotide under conditions that support polymerization of a nascent chain when the added nucleotide is complementary to a base on the target polynucleotide downstream of the primer, wherein the added nucleotide comprises a reporter moiety and a linker, wherein the reporter moiety comprises an electrical charge that is sufficient to result in a detectable change in the resistance of the nanowire, and the reporter moiety is configured to protrude out from the nascent chain so as to reach-down toward the nanowire; and
detecting, upon addition of said nucleotide, the change in the resistance of the nanowire, wherein the change in the resistance is characteristic of the nucleotide added to the nascent chain.
2. The method of claim 1 , wherein the reporter moiety is configured to be removable.
3. The method of claim 2 , wherein the reporter moiety is removed from the added nucleotide after detecting the signal.
4. The method of claim 1 , wherein the reporter moiety is configured not to affect polymerization of the nascent chain by the polymerase.
5. The method of claim 1 , wherein the reporter moiety comprises an aromatic and/or aliphatic skeleton comprising one or more of a tertiary amino group, an alcohol hydroxyl group, a phenolic hydroxy group, or any combinations thereof.
6. The method of claim 1 , wherein the reporter moiety comprises one or more of the following groups or derivatives thereof:
7. The method of claim 1 , wherein the linker comprises a molecule of the following general formula:
H 2 N-L-NH 2
wherein L comprises a linear or branched chain comprising an alkyl group, an oxy alkyl group, or a combination thereof.
8. The method of claim 7 , wherein L comprises a linear chain comprising an alkyl group, an oxy alkyl group, or a combination thereof.
9. The method of claim 8 , wherein a number of carbon atoms in the linear chain is 1 to 100.
10. The method of claim 1 , wherein the added nucleotide further comprises a cleavable cap molecule at the 5′ phosphate group so that addition of another nucleotide is prevented until the cleavable cap is removed.
11. The method of claim 1 , wherein the linker is bound to the 5′ phosphate group of the added nucleotide, thereby acting as a cap.
12. The method of claim 1 , wherein more than one nucleotides are added to the nascent chain, but wherein a successive nucleotide is not added to the nascent chain until after the change in the resistance of the nanowire that is characteristic of the preceding nucleotide added to the nascent chain is detected.
13. The method of claim 1 , wherein the target polynucleotide and the primer comprise molecules selected from the group consisting of DNA, RNA, peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), synthetic nucleotide polymer, and derivatives thereof.
14. The method of claim 1 , wherein the added nucleotide comprises a molecule selected from the group consisting of a deoxyribonucleotide, a ribonucleotide, a peptide nucleotide, a morpholino, a locked nucleotide, a glycol nucleotide, a threose nucleotide, a synthetic nucleotide, and derivatives thereof.