Fluorescent reversible 3’-O-blocked dNTPs are key components of short read sequencing technologies such as reversible termination sequencing-by-synthesis (SBS)[1,2].
Such dNTPs have a common structure:
![Figure 1: Fluorescent reversible blocked 3’-O-Azidomethyl-dNTPs with an Azide-containing cleavable linker (CL) are an established combination for reversible termination SBS [1].](/images/ac81846178/nl_sequencing_0925_1.png)
Figure 1: Azido-based fluorescent reversible 3'-O-blocked dNTPs are an established combination for reversible termination SBS[1]. Red arrows: Tris(2-carboxyethyl)phosphine (TCEP) cleavage sites.
A single fluorescent reversible 3’-O-Azidomethyl-blocked dNTP (A) is incorporated at the 3’-end of DNA by a modified 9°N DNA polymerase followed by detection of the fluorescent signal. The 3’-O-Azidomethyl-blocking group and fluorophore are subsequently removed by TCEP which regenerates the natural 3-OH group (B) and thus facilitates incorporation of the next dNTP.
| Label | 3’-O-Azidomethyl-dUTP | 3’-O-Azidomethyl-dCTP | 3’-O-Azidomethyl-dATP | 3’-O-Azidomethyl-dGTP |
|---|---|---|---|---|
| None | 3’-O-Azidomethyl-5-Propargylamino-dUTP | 3’-O-Azidomethyl-5-Propargylamino-dCTP | 3’-O-Azidomethyl-7-Deaza-7-Propargylamino-dATP | 3’-O-Azidomethyl-7-Deaza-7-Propargylamino-dGTP |
| ATTO532 | 3’-O-Azidomethyl-5-PA-CL- dUTP-ATTO532 | 3’-O-Azidomethyl-5-PA-CL- dCTP-ATTO532 | 3’-O-Azidomethyl-7-Deaza-7- PA-CL- dATP-ATTO532 | 3’-O-Azidomethyl-7-Deaza-7- PA-CL- dGTP-ATTO532 |
[1] Bentley et al. (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53.
[2] Chen et al. (2013) The history and advances of reversible terminators used in new generations of sequencing technology. Genomics Proteomics Bioinformatics 11 (1):34.
