TY - JOUR
T1 - A wax-mediated hot-start polymerase chain reaction improves the genotyping of CYP2C9* 1 and * 3 alleles
AU - Suno, Manabu
AU - Awaya, Toshio
AU - Ogawa, Kento
AU - Ohtaki, Ko Ichi
AU - Chiba, Kaoru
AU - Hayase, Nobumasa
AU - Matsubara, Kazuo
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2002
Y1 - 2002
N2 - Cytochrome P4502C9 (CYP2C9) is a polymorphic enzyme which metabolizes a variety of medications. The CYP2C gene families are extensively homologous; in particular, the nucleotide sequences in the areas adjacent to polymorphic sites are > 95% identical. Frequently, primers designed for amplification of CYP2C9 also amplify other CYP2C genes. Additional amplification of the other CYP2C alleles will thus yield an erroneous restriction pattern, resulting in misleading genotype of CYP2C9. Thus, a procedure to specifically amplify CYP2C9 alleles is necessary. In this study, a wax-mediated hot-start polymerase chain reaction (PCR) for the amplification of CYP2C9* 1 and * 3 alleles was tested. When serially diluted template DNA was amplified by the hot-start PCR followed by the agarose electrophoresis without the enzyme digestion, the 165 bp band derived from CYP2C9 sequence was clearly detected without any non-specifically amplified band even from 10 ng DNA template, and the density of this band increased in proportion to the amount of template. On the other hand, when the PCR without the use of wax was performed, the amplification of the allele was irregular. This hot-start PCR gave a significant increase in the sensitivity for the detection of CYP2C9* 1 and * 3 alleles. The performance of the present method was studied with 64 Japanese volunteers. After the enzyme digestion of the products obtained by the wax-mediated hot-start PCR, only specific bands corresponding to CYP2C9* 1 and * 3 alleles were obtained. This PCR method gave the consistent performance and low background. Thus, the wax-mediated hot-start PCR method should be useful in the genotyping of CYP2C9* 1 and * 3, possibly including CYP2C9* 2 allele in the clinical laboratory.
AB - Cytochrome P4502C9 (CYP2C9) is a polymorphic enzyme which metabolizes a variety of medications. The CYP2C gene families are extensively homologous; in particular, the nucleotide sequences in the areas adjacent to polymorphic sites are > 95% identical. Frequently, primers designed for amplification of CYP2C9 also amplify other CYP2C genes. Additional amplification of the other CYP2C alleles will thus yield an erroneous restriction pattern, resulting in misleading genotype of CYP2C9. Thus, a procedure to specifically amplify CYP2C9 alleles is necessary. In this study, a wax-mediated hot-start polymerase chain reaction (PCR) for the amplification of CYP2C9* 1 and * 3 alleles was tested. When serially diluted template DNA was amplified by the hot-start PCR followed by the agarose electrophoresis without the enzyme digestion, the 165 bp band derived from CYP2C9 sequence was clearly detected without any non-specifically amplified band even from 10 ng DNA template, and the density of this band increased in proportion to the amount of template. On the other hand, when the PCR without the use of wax was performed, the amplification of the allele was irregular. This hot-start PCR gave a significant increase in the sensitivity for the detection of CYP2C9* 1 and * 3 alleles. The performance of the present method was studied with 64 Japanese volunteers. After the enzyme digestion of the products obtained by the wax-mediated hot-start PCR, only specific bands corresponding to CYP2C9* 1 and * 3 alleles were obtained. This PCR method gave the consistent performance and low background. Thus, the wax-mediated hot-start PCR method should be useful in the genotyping of CYP2C9* 1 and * 3, possibly including CYP2C9* 2 allele in the clinical laboratory.
KW - CYP2C9
KW - Genotyping
KW - PCR
KW - Wax-mediated
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U2 - 10.3999/jscpt.33.5_215
DO - 10.3999/jscpt.33.5_215
M3 - Article
AN - SCOPUS:0041319079
VL - 33
SP - 215
EP - 218
JO - Japanese Journal of Clinical Pharmacology and Therapeutics
JF - Japanese Journal of Clinical Pharmacology and Therapeutics
SN - 0388-1601
IS - 5
ER -