Rapid and simple detection of hot spot point mutations of epidermal growth factor receptor, BRAF, and NRAS in cancers using the loop-hybrid mobility shift assay.
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.
Matsukuma
Shoichi
S
Division of Molecular Pathology and Genetics, Kanagawa Cancer Center Research Institute, Nakao 1-1-2, Asahi-ku, Yokohama 241-0815, Japan. matsukum@gancen.asahi.yokohama.jp
Yoshihara
Mitsuyo
M
Kasai
Fumio
F
Kato
Akinori
A
Yoshida
Akira
A
Akaike
Makoto
M
Kobayashi
Osamu
O
Nakayama
Haruhiko
H
Sakuma
Yuji
Y
Yoshida
Tsutomu
T
Kameda
Yoichi
Y
Tsuchiya
Eiju
E
Miyagi
Yohei
Y
D000230:Adenocarcinoma / Q000175:diagnosis / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics*; D008969:Molecular Sequence Data; D015689:Oncogene Protein p21(ras) / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D017384:Sequence Deletion; D013964:Thyroid Neoplasms / Q000235:genetics*
D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D015689:Oncogene Protein p21(ras)