A series of dATP and dCTP nucleotide analogs have been synthesized which are modified by attachment of aliphatic linkers containing a functional group to the amino-nitrogen at the hydrogen bonding positions of the bases, that is, at the 6-position of adenine and the 4-position of cytosine. These nucleotides are incorporated into DNA probes by standard nick-translation protocols. DNA probes labeled with biotin derivatives of these nucleotides are effectively hybridized to target DNA sequences and can be detected by a streptavidin and calf intestinal alkaline phosphatase conjugate with a sensitivity (0.25 pg DNA) sufficient for reproducible and rapid detection of single copy genes in a Southern blot of mammalian DNA.Also, a procedure has been developed to allow reprobing of nylon filters that have been hybridized with biotinylated probes and developed with the streptavidin/alkaline phosphatase conjugate and a standard dye system.

For the first time, we present evidence with restriction enzymes HpaII and MspI which indicates that the proviral DNA sequence of avian sarcoma virus is modified by methylation in a nonpermissive rat cell line but not in permissive chicken cells. Some of the endogenous viral sequences in the permissive cells were also methylated. No 5-methylcytosine could be detected in the unintegrated viral DNA.

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