Terminal fluorescently labeled deoxynucleoside hexaphosphates (dN6Ps) have been established as key components of long-read sequencing technologies such as single-molecule real-time sequencing (SMRT).[1-5]
Labeling deoxynucleotides at the terminal phosphate - rather than on a base - allows the fluorescent tag to be released during DNA synthesis, leaving the growing DNA strand unmodified.[6-9] This supports longer, high-quality sequencing reads, as polymerase activity and base pairing remain unaffected in contrast to base labeling.
Figure 1: Terminal fluorescently labeled deoxynucleoside hexaphosphate core structure for efficient enzymatic incorporation. Arrow: DNA Polymerase cleavage site. The DNA polymerase cleaves the α-β-phosphoryl bond during DNA strand extension (phosphodiester bond formation) thereby releasing a labeled pentaphosphate moiety.
| Label | zeta-(6-Aminohexyl)-dT6P | zeta-(6-Aminohexyl)-dC6P | zeta-(6-Aminohexyl)-dA6P | zeta-(6-Aminohexyl)-dG6P |
|---|---|---|---|---|
| None | zeta-(6-Aminohexyl)-dT6P | zeta-(6-Aminohexyl)-dC6P | zeta-(6-Aminohexyl)-dA6P | zeta-(6-Aminohexyl)-dG6P |
| AZDye 555A | zeta-(6-Aminohexyl)-dT6P-AZDye555A | zeta-(6-Aminohexyl)-dC6P-AZDye555A | zeta-(6-Aminohexyl)-dA6P-AZDye555A | zeta-(6-Aminohexyl)-dG6P-AZDye555A |
| AZDye 568 | zeta-(6-Aminohexyl)-dT6P-AZDye568 | zeta-(6-Aminohexyl)-dC6P-AZDye568 | zeta-(6-Aminohexyl)-dA6P-AZDye568 | zeta-(6-Aminohexyl)-dG6P-AZDye568 |
| AZDye 647A | zeta-(6-Aminohexyl)-dT6P-AZDye647A | zeta-(6-Aminohexyl)-dC6P-AZDye647A | zeta-(6-Aminohexyl)-dA6P-AZDye647A | zeta-(6-Aminohexyl)-dG6P-AZDye647A |
| AZDye 660 | zeta-(6-Aminohexyl)-dT6P-AZDye660 | zeta-(6-Aminohexyl)-dC6P-AZDye660 | zeta-(6-Aminohexyl)-dA6P-AZDye660 | zeta-(6-Aminohexyl)-dG6P-AZDye660 |
[1] Eid et al. (2009) Real-Time DNA Sequencing from Single Polymerase Molecules. Science 323:133.
[2] Korlach et al. (2010) Real-Time DNA Sequencing from Single Polymerase Molecules. Methods in Enzymology 472:431.
[3] Fuller et al. (2016) Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array. PNAS 113(19):5233.
[4] Ibach et al. (2009) Sequencing Single DNA Molecules in Real Time. Angew. Chem. Int. Ed. 48:4683.
[5] Flusberg et al. (2010) Direct detection of dnA methylation during single-molecule, real-time sequencing. Nat. Methods 7:461.
[6] Kumar et al. (2005) Terminal phosphate-labeled nucleotides: synthesis, applications, and linker effect on incorporation by DNA polymerases. Nucleosides, Nucleotides, Nucleic Acids24:401.
[7] Sood et al. (2005) Terminal phosphate-labeled nucleotides with improved substrate properties for homogeneous nucleic acid assays. J Am Chem Soc. 127:2394.
[8] Korlach et al. (2008) Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides. PNAS 27:1072.
[9] Ermert et al. (2017) Phosphate-Modified Nucleotides for Monitoring Enzyme Activity. Top Curr. Chem. 375:28.
