The Fluoride Cleavable 2-(Cyanoethoxy)Methyl (CEM) Group as Reversible 3′-O-Terminator for DNA Sequencing-by-Synthesis—Synthesis, Incorporation, and Cleavage

Abstract

A new and promising sequencing technology called sequencing-by-synthesis (SBS) enables fast determination of DNA sequences. 2'-Deoxynucleotides containing the (2-cyanoethoxy)methyl (CEM) group at the 3'-O-position are potential reversible terminators for the SBS technology. Herein we describe the synthesis, the incorporation by several polymerases, and the cleavage of this 3'-O-blocking group using 3'-O-CEM-thymidinyl-5'-O-triphosphate 7 as an example.

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

“…Inspired by the 2‐(cyanoethoxy)methyl (CEM) protecting group that was initially developed for the chemical synthesis of RNA oligonucleotides by solid‐phase synthesis, CEM has been installed at the 3′‐ O ‐position to synthesize the corresponding 3′‐ O ‐CEM‐5′‐ O ‐thymidinetriphosphate reversible terminator . The nucleotide analogue has been used for incorporation tests on an 23‐mer oligonucleotide template (using a 33 P‐labeled primer) of mixed sequence by different DNA polymerases.…”

“…[106,107] Inspired by the 2-(cyanoethoxy)methyl (CEM)p rotecting group [108] that was initially developed for the chemical synthesis of RNA oligonucleotides by solid-phase synthesis, CEM has been installed at the 3'-O-position to synthesize the corresponding 3'-O-CEM-5'-O-thymidinetriphosphate reversible terminator. [109] The nucleotide analogue has been used for incorporationt ests on an 23-mer oligonucleotide template (using a 33 P-labeled primer) of mixed sequence by different DNA polymerases.S ingle nucleotide incorporation is possible with varying terminatione fficiencies, depending on the nature of the polymerase. Quantitative cleavage of the 3'-O-CEMp rotecting group is observed upon performing the reaction by using tetrabutylammonium fluoride( TBAF) in anhydrous THF within 5min at room temperature or 2min at 37 8C.…”

Fluoride‐Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension

“…Some examples are the 3′- O -(2-nitrobenzyl) group investigated by Metzker and co-workers[6] and Welch and Burgess,[8, 11] the 3′- O -allyl group reported by Metzker,[6] Ju,[12] and Kim,[13] or the 3′- O -azidomethyl group, which was used by Ju and co-workers[14, 15] and was also realised in a commercially available device, the Genome Analyzer developed by Illumina/Solexa. [16, 17] Other interesting groups are the 3′- O -NH 2 group from Benner and co-workers,[18] the 3′- O -(2-cyanoethoxy)methyl group reported by us,[19] or some 3′-blocking groups removable under mild reducing or mild acidic conditions reported by Kwiatkowski. [20] The terminators with bulky 3′-modifications exhibited problems with polymerase acceptance.…”