Hum Genet (1988) 80:309-310 © Springer-Verlag 1988 Detection of a rare-cutter RFLP in a CpG-rich island near the cystic fibrosis locus Philip Stanier, Xavier Estivill*, Nicholas Lench, and Robert Williamson Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, University of London, Norfolk Place, London W2 1PG, UK Summary. The probe pCS.7, isolated from an HpaII tiny frag- ments (HTF) island and tightly linked to the mutation causing cystic fibrosis (CF), detects a polymorphism with the rare-cut- ter restriction enzyme BssHII. In a single digest, the resulting restriction fragment length polymorphism (RFLP) cannot be detected by either conventional or pulse-field gel electropho- resis. A double digest with BssHII in conjunction with a six- cutter restriction enzyme that does not recognize a site contain- ing a CpG dinucleotide enables the probe to be used routinely for prenatal diagnosis and carrier exclusion. This strategy can be used to identify polymorphisms in HTF islands. Introduction HpaII tiny fragments (HTF) islands are distributed through- out the genome and are commonly associated with coding se- quences (Bird 1985, 1986). These islands can be detected using rare-cutter restriction enzymes that recognize CpG-rich sequences and that are methylation sensitive. Jumping lib- raries, which contain rare-cutter sites as positional markers, are used increasingly to move from linkage to locus (Poustka and Lehrach 1986). It is useful to select HTF islands as the end points of these jumps (Estivill et al. 1987a). However, it is often necessary to find a polymorphism in the resulting cloned sequences in order to orient the direction of a jump with respect to recombination events in an associated linkage study. Polymorphisms in HTF islands are less com- mon than in the remainder of the genome because islands are undermethylated. When they do occur, such polymorphisms are difficult to detect due to the clustering of restriction sites. We report a strategy that aids polymorphism searches within the HTF islands. cloning for under-methylated sequences (Estivill et al. 1987a). A subclone, pCS.7, recognizes a polymorphic HhaI site (recog- nition sequence GCGC). This region was sequenced (Fig. 1), revealing that the polymorphism is a point mutation within two overlapping HhaI sites, which together constitute a re- striction site for the rare-cutter enzyme BssHII (GCGCGC). This polymorphism had not been detected previously either by a standard polymorphism search or using pulse-field elec- trophoresis. Unlike two other restriction fragment length polymorph- isms (RFLPs) closely linked to the cystic fibrosis (CF) locus [pXV2C/TaqI (Estivill et al. 1987a), pKM19/PstI (Estivill et al. 1987b)] pCS.7/HhaI has rarely been used for prenatal diag- nosis. The allele sizes are small (600-bp/400-bp) and therefore difficult to interpret except on blots of high quality. We have also found that even complete digests can be mistaken for par- tials on ethidium-stained gels due to the high incidence of methylation elsewhere in the genome at HhaI sites. Using BssHII to detect the polymorphism is equally problematic; pulse-field gel electrophoresis would give allele sizes of 200.35-kb and 200@b which cannot be distinguished, while conventional electrophoresis detects the presence or absence of a 0.25-kb fragment (Fig. 2). When a HindIII/BssHII double digest is used, however, fragments of 1.60-kb and 1.35-kb are obtained, which are easy to interpret with standard methods Methods and results A CpG-rich island associated with an expressed gene, int-1 re- lated protein (IRP) (Wainwright et al. 1988) was isolated by *Present address: Hospital de la Santa Creu i Sant Paul, Universitat Autonoma de Barcelona, Servei d'Haematologia, Barcelona 25, Spain Offprint requests to: P. Stanier Fig.la, b. Sequence comparison of the pCS.7/BssHII (HhaI) poly- morphic site. a Absence of site, A1 allele; b presence of site, A2 al- lele. The polymorphie base is marked with an asterisk