Sequencing-by-synthesis: Fluorescent reversible 3’-O-blocked dNTPs

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:

a reversible blocked 3’-OH group at the ribose to stop elongation after sequence-specific incorporation into DNA.
a fluorophore attached to the base via a cleavable linker (CL) for signal detection after incorporation

Especially the combination of a reversible 3’-O Azidomethyl blocking group and an Azide-containing cleavable linker (CL) has been successfully used in commercially available systems^[1] (Fig. 1).

We now offer a set of correspondingly ATTO532-labeled reversible terminator nucleotides (Tab. 1).

Contact us if you are looking for other fluorophores!

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].

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.

Table 1: Overview of modified 3‘-O-Azidomethyl-dNTPs

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

Fluorescent Dyes and Quenchers - Properties and Structures

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Selected References:

[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.