Long, Processive Enzymatic Dna Synthesis Using 100% Dye-Labeled Terminal Phosphate-Linked Nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by φ29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.

[1]  Nicholas J. Turro,et al.  Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators , 2006, Proceedings of the National Academy of Sciences.

[2]  J. Briggs,et al.  Nucleotide modification at the γ-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase , 2005, Nucleic acids research.

[3]  C. Fuller,et al.  TERMINAL PHOSPHATE LABELED NUCLEOTIDES: SYNTHESIS, APPLICATIONS, AND LINKER EFFECT ON INCORPORATION BY DNA POLYMERASES , 2005, Nucleosides, nucleotides & nucleic acids.

[4]  C. Fuller,et al.  Terminal phosphate-labeled nucleotides with improved substrate properties for homogeneous nucleic acid assays. , 2005, Journal of the American Chemical Society.

[5]  L. Loeb,et al.  Incorporation of reporter-labeled nucleotides by DNA polymerases. , 2005, BioTechniques.

[6]  Jay Shendure,et al.  Fluorescent in situ sequencing on polymerase colonies. , 2003, Analytical biochemistry.

[7]  M. Amacker,et al.  Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA. , 2003, Nucleic acids research.

[8]  S. Quake,et al.  Sequence information can be obtained from single DNA molecules , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Turner,et al.  Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations , 2003, Science.

[10]  S. Brakmann,et al.  The Large Fragment of Escherichia coli DNA Polymerase I Can Synthesize DNA Exclusively from Fluorescently Labeled Nucleotides , 2001, Chembiochem : a European journal of chemical biology.

[11]  B. Angerer,et al.  Progress towards single-molecule sequencing: enzymatic synthesis of nucleotide-specifically labeled DNA. , 2001, Journal of biotechnology.

[12]  M. Sekine,et al.  Efficient synthesis of γ-methyl-capped guanosine 5′-triphosphate as a 5′-terminal unique structure of U6 RNA via a new triphosphate bond formation involving activation of methyl phosphorimidazolidate using ZnCl2 as a catalyst in DMF under anhydrous conditions , 1997 .

[13]  L. Blanco,et al.  Primer‐terminus stabilization at the 3′‐5′ exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. , 1996, The EMBO journal.

[14]  A. Waggoner,et al.  Directly labeled DNA probes using fluorescent nucleotides with different length linkers. , 1994, Nucleic acids research.

[15]  L. Yarbrough.  Synthesis and properties of a new fluorescent analog of ATP: adenosine-5'-triphosphoro-gamma-1-(5-sulfonic acid) napthylamidate. , 1978, Biochemical and biophysical research communications.

[16]  V. A. Kurbatov,et al.  General method for the synthesis of ATP gamma‐derivatives , 1976, FEBS letters.

[17]  D. Hoard,et al.  CONVERSION OF MONO- AND OLIGODEOXYRIBONUCLEOTIDES TO 5-TRIPHOSPHATES. , 1965, Journal of the American Chemical Society.

[18]  I. Barthelemy,et al.  In vivo transcription ob bacteriophage ø29 DNA: Transcription initiation sites , 1965 .

[19]  L. Blanco,et al.  Relating structure to function in phi29 DNA polymerase. , 1996, The Journal of biological chemistry.

[20]  J. A. Esteban,et al.  Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization. , 1993, Journal of Biological Chemistry.

[21]  M. Yoshikawa,et al.  A novel method for phosphorylation of nucleosides to 5'-nucleotides. , 1967, Tetrahedron letters.