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Reversible Terminators for Primer Extension

Firebird has introduced the 3'-ONH2 reversible terminator as an alternative to the larger 3'-OCH2N3 groups and their problematic enzymology and cleavage.

 

How do they work?

The 3’-ONH2 group is accepted by a variety of polymerases. After incorporation, further primer extension is blocked. The 3'-ONH2 group is cleaved with buffered aqueous sodium nitrite to regenerate a standard 3'-OH.                   

 

 

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What are they used for?

3’-ONH2 reversible terminators can be used in DNA sequencing (Hutter et al., 2010), Oligonucleotide synthesis (Jensen and Davis, 2018, Sarac and Hollenstein, 2019) and SNP analysis (Chen et al., 2010).

 

Which reversible terminator is right for your application?

Firebird offers reversible terminators in several different formats:

 

     The ONH2-170 series: Ready to use, untagged

     The ONH2-100 series: Oxime blocked, untagged 

     The ONH2-DT series: Diol-linked tags

     The ONH2-DS series: Disulfide-linked tags

 

The 170 series ("ready to use") are crude products from a solid phase synthesis procedure that results in approximately 90% pure terminator triphosphates, which should be sufficient for most purposes. This series carries a free 3'-ONH2 group and can be incorporated by various enzymes without further modification. The shelf life of this series is about one year (if stored properly, at or below -20°C).

 

The 100 series ("oxime blocked") are the acetoxime form of the 170 series and has a longer shelf life (many years, if stored properly). This series is HPLC purified to remove most of the impurities, albeit at an increased cost. But these oximes will not be incorporated by enzymes. For enzymatic incorporation the free 3'-ONH2 must be regenerated before use, which can be achieved in situ with buffered aqueous methoxylamine.

 

The DT and DS series are reversible terminators carrying a cleavable linker attached to the nucleobase with a free amino group, allowing for the attachment of a tag (e.g. fluorescent dye or biotin). As in the 100 series, the 3'-ONH2 group is protected as the acetoxime, for attachment of the tag and subsequent purification as well as increased shelf life. And as with the 100 series, this oxime needs to be deprotected with buffered aqueous methoxylamine before use. Upon incorporation of the terminator into an oligonucleotide the linker can be cleaved under mild conditions (dilute aqueous periodate for the DT series, phosphine or thiol reagents for the DS series) to remove the tag.

 

For a fee, Firebird scientists can attach a customer specified tag onto the primary amine; please inquire for pricing.