(Chemical Equation Presented) Serving a double purpose: 3′-O-fluorophore-labeled dTTPs were synthesized and used as reversible terminators of DNA polymerization. Successful incorporation of nucleotides with a bulky 3′-O-fluorophore and detection of fluorescence for base calling can potentially be used to improve sequencing-by-synthesis technology.
"Within the scope of this study, several 3′-blocking groups were investigated, including bulky esters and ethers with the label attached to the blocking group and small groups. Some examples are the 3′-O-(2-nitrobenzyl) group investigated by Metzker and co-workers and Welch and Burgess,[8, 11] the 3′-O-allyl group reported by Metzker, Ju, and Kim, 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-NH2 group from Benner and co-workers, the 3′-O-(2-cyanoethoxy)methyl group reported by us, or some 3′-blocking groups removable under mild reducing or mild acidic conditions reported by Kwiatkowski. "
[Show abstract][Hide abstract] ABSTRACT: Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3'-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3'-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.
Chemistry - A European Journal 03/2011; 17(10):2903-15. DOI:10.1002/chem.201001952 · 5.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In recent years, the use of genome-wide technologies has revolutionized the study of eukaryotic transcription producing results for thousands of genes at every step of mRNA life. The statistical analyses of the results for a single condition, different conditions, different transcription stages, or even between different techniques, is outlining a totally new landscape of the eukaryotic transcription process. Although most studies have been conducted in the yeast Saccharomyces cerevisiae as a model cell, others have also focused on higher eukaryotes, which can also be comparatively analyzed. The picture which emerges is that transcription is a more variable process than initially suspected, with large differences between genes at each stage of the process, from initiation to mRNA degradation, but with striking similarities for functionally related genes, indicating that all steps are coordinately regulated. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
[Show abstract][Hide abstract] ABSTRACT: The eukaryotic RNA polymerase II (RNAPII) catalyzes the transcription of all protein encoding genes and is also responsible for the generation of small regulatory RNAs. RNAPII has evolved a unique domain composed of heptapeptide repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the C-terminus (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation patterns of serine residues in CTD during gene transcription coordinate the recruitment of factors to the elongating RNAPII and to the nascent transcript. Recent studies identified threonine 4 and tyrosine 1 as new CTD modifications and thereby expanded the "CTD code". In this review, we focus on CTD phosphorylation and its function in the RNAPII transcription cycle. We also discuss in detail the limitations of the phosphospecific CTD antibodies, which are used in all studies. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.