Labeled Nucleoside Triphosphates with Reversibly Terminating Aminoalkoxyl Groups

Abstract

Nucleoside triphosphates having a 3′-ONH₂ blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3′-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3′-ONH₂ group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3′-ONH₂ blocking group in “next generation sequencing.”

Keywords:

• triphosphate
• aminooxy
• mutant polymerases
• sequencing technology
• fluorescent nucleotide
• oligonucleotide microarray
• nitrous acid
• primer elongation
• alpha effect
• synthesis
• oxime

Acknowledgments

The Foundation and Firebird were supported by grants from the NIGMS (2R42HG003668-02A) and NHGRI (R41HG004589), as well as by Nucleic Acids Licensing, LLC.

Supplemental material is available that includes the synthetic procedures and characterization of all synthesized compounds, various primer extension experiments and sequencing cycles in solution using these triphosphates and various polymerases, and more detailed procedures describing the sequencing runs performed on the Intelligent Bio-Systems Instrument.

Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides and Nucleic Acids to view the free supplemental file.