One-Tube Restriction Enzyme Digest and Fluorescent Labeling for Restriction Endonuclease Fingerprinting Single-Strand Conformational Polymorphism

Abstract

at the end of the PCR product and not randomly distributed within the PCR product. We have designed a fluores cence REF-SSCP protocol for mutation or SNP detection by combining widely used technologies (i.e., restriction digest, DNA minisequencing, and SSCP analysis) and have tested it by performing a blind test on a collection of mutated cDNAs of human phenyl alanine hydroxylase (PAH). When performing the blind test, we detected gel shifts in all mutated samples. Following PCR amplification, the PCR product (1544 bp) was digested