Nucleic Acids Research, Vol 27, Issue 2 389-395, Copyright © 1999 by Oxford University Press
D Chen, Z Yan, DL Cole and GS Srivatsa
The purity of a drug substance can influence its toxicity and potency, so
impurities must be specifically determined. In the case of synthetic
oligodeoxyribonucleotide drugs, however, product complexity makes complete
impurity speciation difficult. The goal of the present work was to develop
a new analytical method for speciation of individual internal (n-1)mer
impurities arising from formal nucleotide deletion in synthetic
oligodeoxyribonucleotides. A complete series of oligodeoxyribonucleotide
probes were designed, each complementary to an (n-1)mer deletion sequence
of the drug in question. Glass plates were used as a solid support for
individually immobilizing the entire probe array. The total mixture of
internal (n-1) length impurities was isolated from a synthetic
oligodeoxyribonucleotide by PAGE and labeled with 35S. Under stringently
optimized conditions, only the perfectly sequence-matched
oligodeoxyribonucleotide hybridized to each probe, while all other deletion
sequences were removed by washing with buffer. The 35S signal intensity of
the bound oligodeoxyribonucleotide was proportional to the concentration of
each (n-1)mer deletion sequence in the analyte solution. This method has
been applied to a number of synthetic phosphorothioate
oligodeoxy-ribonucleotide lots and shown to be reliable for speciation and
relative quantitation of the internal (n -1)mer deletion sequences present.
ARTICLES
Analysis of internal (n-1)mer deletion sequences in synthetic oligodeoxyribonucleotides by hybridization to an immobilized probe array
Department of Development Chemistry, Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008, USA. dchen@isisph.com
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