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Positional cloning relies on closely spaced regional DNA markers. Here, we report on the combined use of preparative pulsed-field gel electrophoresis (PFGE) and interspersed repetitive sequence (IRS) polymerase chain reaction (PCR) amplification to isolate two DNA markers from a 660 kb Sfil fragment that contains locus DXS14 and maps to a region between disease-specific incontinentia pigmenti type 1 (IP1) chromosome translocation breakpoints (1). Genomic DNA from a somatic cell hybrid containing a human X chromosome (GM06318) and a radiation hybrid (RH 128) containing approximately 10% of a human X chromosome, including DNA markers spanning the IP1 X-chromosomal translocation breakpoints within Xpll.21 (2), was digested with Sfil and fractionated by PFGE. By Southern blot analysis, probe p58.1 (DXS14) and probe 30CE2.8 (DXS370) detected a 660 kb and a 400 kb fragment, respectively, in both hybrid DNA samples (Figure 1). Sequential hybridizations of the same membrane with human and hamster genomic DNA indicated that most human and hamster Sfil fragments ranged from 250 kb to

nucleic acids research

Interspersed repetitive sequence (IRS)-PCR amplification of pulsed-field gel fractionated DNA to derive markers from the incontinentia pigmenti 1 (IP1) locus