A simple and rapid method to detect the
epidermal growth factor receptor hot spot mutation L858R in
lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded
oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the
oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated
DNA was distinguishable in a native
polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of
epidermal growth factor receptor in
lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type
Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in
thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant
DNA comprising 7.5% of the total
DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical
cancers. Other applications are also discussed.