Quantitative analysis of carbodiimide modified DNA and immunoprobing by adduct specific antibodies.
Antibodies have been raised against N-cyclohexyl-N-(4-methylmorpholinium)ethyl carbodiimide (CMC) modified single-stranded DNA and characterized by competitive and non-competitive immunoassays to be highly specific for CMC base adduct in homopolymers poly(dG), poly(dT) and DNA. The antibodies recognize picogram concentrations of CMC treated DNA with no cross reactivity to at least 1000-fold excess of unmodified DNA or CMC treated poly(dA). The detection limit of antibodies at 1.4 fmol CMC adduct allows quantitation at a CMC/base ratio of 4.6.10(-7). Based upon single modified base-containing synthetic oligomers, a 7-fold higher binding preference is observed for CMC modified thymine than guanine bases. CMC binding to supercoiled DNA is found to depend upon reaction temperature and ionic strength. CMC-modified supercoiled SV40 and ColE1 DNA, exhibit specific antibody binding proportional to the DNA concentration and extent of CMC modification. However, antibody binding observed is independent of the conformation or strandedness of CMC-modified DNA. DNA extensively modified with CMC retains its inherent capacity to specifically and quantitatively hybridize with complementary DNA immobilized to membranes upon direct blotting or Southern transfers from gels. Hybridized CMC-DNA, through antibody binding, provides for the sensitive and non-isotopic detection of the target DNA sequences.
Wani
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Department of Radiology, Ohio State University, Columbus.
Yamasaki
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EF
D000915:Antibody Affinity; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D015139:Blotting, Southern; D003028:CME-Carbodiimide / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D004278:DNA, Superhelical / Q000737:chemistry / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011089:Polydeoxyribonucleotides / Q000737:chemistry* / Q000276:immunology; D015183:Restriction Mapping; D013329:Structure-Activity Relationship
D000936:Antigen-Antibody Complex; D004278:DNA, Superhelical; D015345:Oligonucleotide Probes; D011089:Polydeoxyribonucleotides; D003028:CME-Carbodiimide; D004247:DNA