Modifications of guanine bases during oligonucleotide synthesis.
Guanine bases are sensitive to modification during automated DNA synthesis and processing reactions. Methods for the detection of two types of guanine modifications are described. The first method uses the higher reactivity of the modified G base to KMn04 oxidation than T bases, and thus allows detection by chemical DNA sequencing. The second method makes use of the Escherichia coli nucleotide excision repair enzyme UvrABC endonuclease which can detect "bulky" base modifications at each nucleotide in the synthetic DNA. Though the chemical structures of the two modifications are not known, they may be related. Both types of G modifications are often found in oligonucleotides synthesized by the methoxy-diisopropyl-phosphoramidite (MEDP) chemistry but non-detectable in the products of the beta-cyanoethyl-diisopropyl-phosphoramidite (CEDP) chemistry. The Rubin and Schmid pyrimidine-specific chemical DNA sequencing procedure (Rubin, C.M., and Schmid, C.W. (1980) Nucleic Acids Res. 8, 4613-4619) was found to be applicable to oligonucleotides synthesized by the CEDP chemistry, and to oligonucleotides synthesized by the MEDP chemistry if precautionary measures are taken to destroy the signals produced by the highly KMnO4 sensitive modified guanine bases. We also show how chemical DNA sequencing might be useful for diagnosing other chemical modifications in synthetic oligonucleotides.
Yeung
A T
AT
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111.
Dinehart
W J
WJ
Jones
B K
BK
D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis*; D004706:Endodeoxyribonucleases; D029968:Escherichia coli Proteins; D006147:Guanine; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*
D029968:Escherichia coli Proteins; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D006147:Guanine; D004247:DNA; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC