Restriction Endonuclease Fingerprinting (REF): A Sensitive Method for Screening Mutations in Long, Contiguous Segments of DNA

摘要

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 factorIX gene was amplified with PCR and digested with each of five groups of restriction endonucleases. The endonu-cleases 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 kina.se, 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~(TM) at temperatures of either 23° or 8°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 restrictioncomponent 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 singlestranded segment of the SSCP component for the 24 point mutations ranged from 49% for polyacrylamide at 23°C to 68% with GeneAmp at 8°C. REF detected 96% of the mutations with polyacrylamide at 23°C and 100% with GeneAmp at 23° or 8°C. GeneAmp at 23° and 8°C also detected 100% of a subsequent blinded sample that contained normal controls and 27 different point mutations The data suggest that REF is a highly sensitive and specific method in which 50~+ kb of sequence (1~+ kb x .50 lanes) can be screened with a single gel.