Restriction endonuclease fingerprinting (REF) is a modification of single-strand confirmation polymorphism (SSCP) that was developed to detect the presence of essentially all mutations in a 1-kb segment. To test REF, a 1-kb segment of the human factor IX gene was amplified with PCR and digested with each of five groups of restriction endonucleases. The endonucleases in each group were chosen so that the average size of the fragments was about 150 bp. After separate digestions, the products were mixed, 5' end-labeled with T4 polynucleotide kinase, denatured and electrophoresed under nondenaturing conditions. Each lane screened 1 kb and typically contained 68 segments (6.8 fragments per average digestion x 5 digestions x 2 strands). REF was performed with 5.6% polyacrylamide and 7.5% GeneAmp at temperatures of either 23 degrees or 8 degrees C. Point mutations resulted in the gain or loss of a restriction site in 21% of 24 test mutations (informative restriction component). In cases in which the restriction component was not informative, mutations were detected if any of the five mutation-containing restriction fragments (producing 10 single-stranded segments) displayed abnormal mobility (SSCP component). The average efficiency per single-stranded segment of the SSCP component for the 24 point mutations ranged from 49% for polyacrylamide at 23 degrees C to 68% with GeneAmp at 8 degrees C. REF detected 96% of the mutations with polyacrylamide at 23 degrees C and 100% with GeneAmp at 23 degrees or 8 degrees C. GeneAmp at 23 degrees and 8 degrees C also detected 100% of a subsequent blinded sample that contained normal controls and 27 different point mutations.(ABSTRACT TRUNCATED AT 250 WORDS)