Four-color DNA sequencing with 3′- O -modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides

Abstract

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction can decipher many sequences in parallel. We report here a DNA sequencing method that is a hybrid between the Sanger dideoxynucleotide terminating reaction and SBS. In this approach, four nucleotides, modified as reversible terminators by capping the 3-OH with a small reversible moiety so that they are still recognized by DNA polymerase as substrates, are combined with four cleavable fluorescent dideoxynucleotides to perform SBS. The ratio of the two sets of nucleotides is adjusted as the extension cycles proceed. Sequences are determined by the unique fluorescence emission of each fluorophore on the DNA products terminated by ddNTPs. On removing the 3-OH capping group from the DNA products generated by incorporating the 3-O-modified dNTPs and the fluorophore from the DNA products terminated with the ddNTPs, the polymerase reaction reinitiates to continue the sequence determination. By using an azidomethyl group as a chemically reversible capping moiety in the 3-O-modified dNTPs, and an azido-based cleavable linker to attach the fluorophores to the ddNTPs, we synthesized four 3-O-azidomethyl-dNTPs and four ddNTP-azidolinker-fluorophores for the hybrid SBS. After sequence determination by fluorescence imaging, the 3-O-azidomethyl group and the fluorophore attached to the DNA extension product via the azidolinker are efficiently removed by using Tris(2-carboxyethyl)phosphine in aqueous solution that is compatible with DNA. Various DNA templates, including those with homopolymer regions, were accurately sequenced with a read length of >30 bases by using this hybrid SBS method on a chip and a four-color fluorescence scanner.sequencing by synthesis ͉ DNA chip T he completion of the Human Genome Project (1) was a monumental achievement in biological science. The engine behind this project was the Sanger sequencing method (2), which is still the gold standard in genome research. The prolonged success of the Sanger sequencing method is because of its efficiency and fidelity in producing dideoxy-terminated DNA products that can be separated electrophoretically and detected by fluorescence (3-5). However, a challenge in the use of electrophoresis for DNA separation is the difficulty in achieving high throughput and the complexity involved in the automation, although some level of increased parallelization may be achieved by using miniaturization (6).To overcome the limitations of the Sanger sequencing technology, a variety of new methods have been investigated. Such approaches include sequencing by hybridization (7), mass spectrometry sequencing (8, 9), sequencing by nanopores (10), and sequencing by ligation (11). More recently, DNA sequencing by synthesis (SBS) approaches such as pyrosequencing (12), sequencing of single DNA molecules (13,14), and polymerase colonies (15) have been widely explored. Previously, we reported the development of a general strategy to rationally design cleavable fluorescent nucleotide reversible terminators (NRTs) ...

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

“…These include next-generation sequencing approaches (1)(2)(3), single molecule sequencing (4), labeling of DNA and PCR amplificates, e.g., for microarray analysis (5)(6)(7)(8), DNA conjugation (9), or the in vitro selection of ligands such as aptamers by SELEX (systematic enrichment of ligands by exponential amplification) (10). Furthermore, utilizing the intrinsic properties of DNA in combination with chemically introduced functionalities provides an entry to new classes of nucleic acids-based hybrid materials (9).…”

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

“…We recently demonstrated proof-of-principle for the nanopore SBS sequencing method (11), which combines SBS (18)(19)(20) with nanopore-based identification of different-sized polymer tags (8,9) attached to the nucleotides. One of four different-length PEG tags was attached to the terminal phosphate of each nucleotide.…”

RNA Cloaking by Reversible Acylation

“…Azide reduction with phosphines has been studied for multiple biological and biotechnological applications: for example, for reduction of azidophenylalanine in proteins, [19] and as a reversible terminator in sequencing chemistry. [7] O-azidomethylbenzoyl groups were previously used in organic synthesis to protect alcohols and amines, and were released during the deprotection step with aryl phosphines. [20] To test our proposed cloaking-uncloaking strategy with NAI-N 3 , we chose to study reactions initially with short transfer RNA-derived RNA fragments (tRFs).…”

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