A chemical method for introducing haptens onto DNA probes.
We have developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. We have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 X 10(-17) mol (1 X 10(6) copies) of HBV DNA in total DNA from human serum.
Keller
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Biotech Research Laboratories, Inc., Rockville, Maryland 20850.
Cumming
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Huang
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Manak
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Ting
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R
D019297:2,4-Dinitrophenol; D000345:Affinity Labels; D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004140:Dinitrophenols / Q000032:analysis / Q000276:immunology; D006241:Haptens; D006515:Hepatitis B virus / Q000235:genetics; D006801:Humans; D009693:Nucleic Acid Hybridization; D011817:Rabbits; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics
D000345:Affinity Labels; D004140:Dinitrophenols; D006241:Haptens; D014764:Viral Proteins; D004247:DNA; D019297:2,4-Dinitrophenol