Terminal fluorescently labeled dN6Ps - Key components of long-read sequencing technologies

JENA BIOSCIENCE NEWS

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.

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