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

Effects of the formation of the multistranded structure on electrophoretic mobility. DNA fragments (pSP64 DNA) with various sizes (300, 500, 900, and 1200 bp), were subjected to direct marking using a labeled probing oligonucleotide (probe 4; 60 bp) and a splint oligonucleotide (17 bp). As controls, the target DNA fragments were ³²P-labeled at the 5′-termini by T4 polynucleotide kinase and elecrophoresed in parallel. (Lanes 1,3,5,7) Results of autoradiographic patterns after direct marking; (lanes 2,4,6,8) ³²P-labeled target DNA fragments (indicated by arrowheads). (Lanes 1,2) 300-bp DNA fragment; (lanes 3,4) 500-bp DNA fragment; (lanes 5,6) 900-bp DNA fragment; (lanes 7,8) 1200-bp DNA fragment. For details, see Methods.