Marking of specific sequences in double-stranded DNA molecules—SNP detection and direct observation

Direct marking of specific sequences in human genomic DNA. Human genomic DNA (digested with EcoRI, HindIII, and PvuII) were subjected to direct marking using a labeled probing oligonucleotide and a splint oligonucleotide (17 bp). (A) A diagrammatic representation of 1909- and 3159-bp DNA fragments in p53 gene. Probe 7a and probe 8a cover 40-bp sequences, respectively. (B) Autoradiographic (left) and ethidium bromide staining (right) patterns after direct marking. (Lane 1) Probed by probe 7a; (lane 2) probed by probe 8a. (C) Autoradiographic (left) and ethidium bromide staining (right) patterns after Southern hybridization. (Lane 1) Probed by probe 7b; (lane 2) probed by probe 8b. For probe 7b and 8b, we used a ³²P-labeled DNA fragment produced by PCR with forward primer 6 and backward primer 5 (for probe 7b), forward primer 7 and backward primer 6 (for probe 8b) as described the Methods. Nucleotide numbers (nt. numbers) are those registered in GenBank (accession no. X54156). Distances in the diagram do not reflect actual distances. For details, see Methods.