Partitioned pulsed-field gel electrophoresis-PCR (PPF-PCR): a new method for pulsed-field mapping for STS and microsatellites.

Pulsed field gel electrophoresis (PFGE) is an important tool in the construction of physical maps of genomic DNA. The colocalization of probe DNA to a large restriction enzyme generated fragment demonstrates the contiguity of the selected markers. At present, the Southern blot and its subsequent hybridization are the only practical means to colocalize probes on a PFGE restriction. This method is time consuming and especially difficult when the hybridization probe contains repeat sequences. Two very important classes of probes are effectively excluded from direct use in probing PFGE: [1] Sequence Tagged Sites (STS) which yield PCR generated probes whose small size often causes a poor hybridization signal, and [2] Microsatellite repeats (MS), which are at the core of the largest class of genomic markers, yield PCR generated probes that are likewise small but also highly cross-reactive due to the core of tandem repeats. We present here a new non-radioactive method based on PCR that can incorporate these markers. We call this new technique Partitioned Pulse Field Gel Electrophoresis-PCR or PPF-PCR. To test this method we have applied it to the previously well mapped region of Charcot—Marie—Tooth disease type 1A, localized to chromosome 17pl 1.2 (1,2).