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Original Articles

NOVEL NUCLEOSIDE TRIPHOSPHATE ANALOGS FOR THE ENZYMATIC LABELING OF NUCLEIC ACIDS

, , , , &
Pages 1141-1145 | Published online: 07 Feb 2007

REFERENCES

  • Lipshutz , R. J. , Fodor , S. , Gingeras , T. R. and Lockhart , D. J. 1999 . Nature Genet. , 21 : 20 – 24 . and references cited therein
  • McGall , G. H. 1997 . Biochip Arrays and Integrated Devices for Clinical Diagnostics Edited by: Hori , H. International Business Communications Library Series . Chapter 2, and references cited therein
  • “ The synthesis of these analogs is too lengthy to appear here, and will be described in a separate paper ” . In All spectral characterization data were consistent with the assigned structure of intermediates and the final nucleoside triphosphates
  • “ The incorporation efficiency for TdT labeling of d(pT)16 was determined by IE-HPLC analysis, from the ratio of peak areas of the unlabeled and labeled oligonucleotide (absorbance detection at 260 nm) ” . In For IVT labeling of RNA using T7 RNA polymerase, a 1.2 kb transcript from a crude IVT reaction was resolved from unincorporated nucleotides by IE-HPLC, and the incorporation of the fluorescein was determined from peak area ratios by simultaneous absorbance detection of the RNA transcript at 260 nm and 495 nm
  • Pochet , S. , Dugue' , L. , Meiera , A and Marlie're , P. 1995 . The desired N1-β isomer was identified by comparison of NMR chemical shift and NOE data with that reported for related nucleosides . Biorg. Med. Chem. Lett. , 5 : 1679 – 1684 .
  • Commercially available from NEN
  • Labeling reaction conditions were not optimized
  • Prepared from formycin A, following procedures similar to those reported in reference 10
  • et. al. , K. 1996 . Prepared from β-D-ribofuranosyl-2,4(1H,3H)-pyrimidinedione, according to Muhlegger WO 96/28640
  • These values could not be determined reliably because of competing disproportionation activity at higher enzyme concentrations
  • The labeling of target DNA followed the recommended procedure for the commercially available assay, except that 4-times the amount of TdT (100 u) was used to that for the control (25 u)
  • The labeling of RNA target with 3b followed the recommended procedure for the commercially available assay, except that the UTP:analog triphosphate ratio was decreased from 1:2 to 1:5

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