Labeling deoyxnucleotides at the terminal phosphate - rather than on the base - enables the fluorescent tag to be released during DNA synthesis (Fig. 1)[1-4]. This leaves the growing DNA strand unmodified, supporting longer, high-quality sequencing reads since polymerase processivity and DNA base pairing properties are not affected by a remaining label.
Extension of the phosphate chain from triphosphate to tetra-, penta- or hexaphosphate strongly increases the enzymatic incorporation efficiency[2-4]. Especially terminal fluorescently labeled deoxynucleoside hexaphosphates (dN6Ps) (Fig. 1) in combination with Phi29 DNA polymerase have been established as key components of long-read sequencing technologies such as single-molecule real-time sequencing (SMRT)[5-9].
Figure 1: Terminal fluorescently labeled deoxynucleoside hexaphosphate (dN6P) 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] Ermert et al. (2017) Phosphate-Modified Nucleotides for Monitoring Enzyme Activity. Top Curr. Chem. 375:28.
[2] Kumar et al. (2005) Terminal phosphate-labeled nucleotides: synthesis, applications, and linker effect on incorporation by DNA polymerases. Nucleosides, Nucleotides, Nucleic Acids 24:401.
[3] Sood et al. (2005) Terminal phosphate-labeled nucleotides with improved substrate properties for homogeneous nucleic acid assays. J. Am. Chem. Soc. 127:2394.
[4] Korlach et al. (2008) Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides. PNAS 27:1072.
[5] Eid et al. (2009) Real-Time DNA Sequencing from Single Polymerase Molecules. Science 323:133.
[6] Korlach et al. (2010) Real-Time DNA Sequencing from Single Polymerase Molecules. Methods in Enzymology 472:431.
[7] Ibach et al. (2009) Sequencing Single DNA Molecules in Real Time. Angew. Chem. Int. Ed. 48:4683.
[8] Flusberg et al. (2010) Sequencing Single DNA Molecules in Real Time. Nat. Methods 7:461.
[9] Fuller et al. (2016) Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array. PNAS 13(19):5233.
