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In a blinded analysis, restriction endonuclease fingerprinting detects all the mutations in a 1.9-kb segment.

Single-strand conformation polymorphism (SSCP) (4) is the most widely used DNA screening method. SSCP detects single-base sequence changes by abnormal electrophoretic migration of one or both single strands on a nondenaturing gel. The method does not detect all sequence changes, and the sensitivity is a complex function of sequence context and size (1). Dideoxy fingerprinting (ddF) and bi-directional dideoxy fingerprinting (bi-ddF) were developed as hybrids of SSCP and either conventional Sanger dideoxy sequencing or bidirectional cycle sequencing. DdF and bi-ddF generate substantial redundancy in the SSCP effect, thereby detecting virtually 100% of single-base changes in segments of <300 bp and 300–600 bp, respectively (2,6). Restriction endonuclease fingerprinting (REF) is another hybrid method, which can detect virtually all mutations in a 1-kb segment (3). We show that twice as much sequence can be screened with REF (2 kb from 50 samples in a typical manual gel) with a small increase in effort. In addition, we provide evidence that REF is both a workhorse technique and can be applied to genes with GC contents of 70%. For REF, a DNA segment is amplified by polymerase chain reaction (PCR), digested separately with different restriction enzymes, mixed, labeled, denatured and electrophoresed on a nondenaturing gel. Previously, five different restriction endonuclease digestions were performed in a blinded analysis that detected all of 51 mutations in a 1-kb segment (3). We use six groups of restriction endonucleases to analyze a 1.94-kb segment of the human factor IX gene. Thus, any sequence change will be present in six restriction segments that yield twelve single-stranded segments (unless the mutation creates or destroys one or more of the restriction endonuclease sites). A change in mobility in any one of these segments is sufficient to detect the presence of a mutation. A change in mobility might reflect either a change in secondary structure (the SSCP component of REF) or an altered restriction