CAJ | 학술논문

目的建立单管双向荧光PCR方法快速测定人NAD(P)H:醌氧化还原酶1[NAD(P)H:quinone oxidoreductase 1,NQO1]基因609C/T多态性.方法以人NQO1基因中的609C/T位点,设计双向引物,优化反应条件,应用SYBR GreenⅠ双向荧光PCR扩增191份人基因组DNA标本,并通过对产物进行熔解曲线分析,根据产物Tm值进行等位基因单核苷酸多态性分型.对其中62份标本用经典的PCR-限制性片段长度多态方法进行基因型分型,验证结果准确性.结果 62份样本的单管双向荧光PCR方法的基因型结果与PCR-限制性片段长度多态法分型结果符合率100%.191份样本中,纯合野生型(CC)占28%,杂合型(CT)占50%,纯合突变型(TT)占22%.结论单管双向荧光PCR方法检测NQO1基因609C/T多态性,操作简便、反应过程快速,结果直观,敏感性、准确性和稳定性好,适用于临床样本检测及流行病学调查研究.
목적 건립단관쌍향형광PCR방법쾌속측정인NAD(P)H:곤양화환원매1[NAD(P)H:quinone oxidoreductase 1,NQO1]기인609C/T다태성.방법 이인NQO1기인중적609C/T위점,설계쌍향인물,우화반응조건,응용SYBR GreenⅠ쌍향형광PCR확증191빈인기인조DNA표본,병통과대산물진행용해곡선분석,근거산물Tm치진행등위기인단핵감산다태성분형.대기중62빈표본용경전적PCR-한제성편단장도다태방법진행기인형분형,험증결과준학성.결과 62빈양본적단관쌍향형광PCR방법적기인형결과여PCR-한제성편단장도다태법분형결과부합솔100%.191빈양본중,순합야생형(CC)점28%,잡합형(CT)점50%,순합돌변형(TT)점22%.결론 단관쌍향형광PCR방법검측NQO1기인609C/T다태성,조작간편、반응과정쾌속,결과직관,민감성、준학성화은정성호,괄용우림상양본검측급류행병학조사연구.
Objective To develop a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NAD(P)H:quinone oxidoreductase 1(NQO1)gene.Methods Two primers of NQO1 gene C609T locus were designed.Using these primers,a SYBR Green Ⅰ fluorescent bidirectional PCR,combined with melting curve analysis of the PCR products,were optimized to differentiate the 609C/T polymorphisms in 191 samples of human genomic DNA.The accuracy of the fluorescent bidirectional PCR was validated by the classical method of PCR-restriction fragment length polymorphism(RFLP)in 62 of these 191 samples.Results In the 62 samples,the genotypes determined by the fluorescent bidirectional PCR were 100%consistent with the ones by the PCR-RFLP.The frequencies of genotypes of homozygous wild-type(CC),heterozygous(CT),and homozygous mutant(TT)were 28%,50%,and 22%,respectively,in the 191 samples.Conclusion The single-tube fluorescent bidirectional PCR method established here provides a simple,rapid,accurate and inexpensive assay to determine the 609C/T pelymorphism of NQO1 gene.The assay is suitable to detect the single nucleotide polymorphism in large-scale samples.