Simple and accurate determination of CYP2D6 gene copy number by a loop-mediated isothermal amplification method and an electrochemical DNA chip.
BACKGROUND
A combination technology of a loop-mediated isothermal amplification (LAMP) method and an electrochemical DNA chip has been developed. In this study, the CYP2D6 gene copies were detected by this technology for determination of the functional CYP2D6*5 and CYP2D6*2x2 alleles.


METHODS
A set of LAMP primers was designed to coamplify the CYP2D6 gene, but not the CYP2D6*36, and the CYP2D8P gene, which would enable determination of the CYP2D6 gene copies by relatively comparing with the amount of the amplified products of CYP2D6 and CYP2D8P. The LAMP products were reacted with the electrochemical DNA chip using the DNA detection system Genelyzer that automatically controls hybridization reaction, washing, and electrochemical detection. To test the feasibility of the system, 16 samples that have various combinations of copy numbers were selected from pooled samples previously genotyped according to empirically well-authorized Southern blotting-based RFLP methods.


RESULTS
The CYP2D6 gene copies were consistent with the previous genotypes except a rare CYP2D6*18 allele probably due to mutation near the primer region. The results were completely reproducible in a blind test and were given within 1.5h.


CONCLUSIONS
This method offers a simple and accurate determination of the CYP2D6 gene copies and is expected to contribute to personalized medicine.
Nakamura
Naoko
N
Corporate Research & Development Center, Toshiba Corporation, Komukai-Toshiba-cho, Saiwai-ku, Kawasaki, Kanagawa, Japan. nao.nakamura@toshiba.co.jp
Fukuda
Tsuyoshi
T
Nonen
Shinpei
S
Hashimoto
Koji
K
Azuma
Junichi
J
Gemma
Nobuhiro
N
D000483:Alleles; D001483:Base Sequence; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004563:Electrochemistry; D018628:Gene Dosage / Q000235:genetics*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods
D004247:DNA; D019389:Cytochrome P-450 CYP2D6