摘要
目的 比较Taqman荧光定量PCR法、SYBR GreenⅠ荧光定量PCR法和普通PCR法制作3-磷酸甘油醛（GAPDH）标准曲线的检出下限、线性范围的差异，确定一种较好的GAPDH绝对定量方法。方法 构建含GAPDH全长的质粒，经PCR和EcoRⅠ限制性酶切鉴定确认后，紫外定量并连续稀释10倍作为标准品。分别用Taqman法和SYBR GreenⅠ法于荧光定量PCR仪上制作标准曲线，同时用普通PCR结合琼脂糖凝胶电泳利用Quantity One软件定量并制作标准曲线。结果 Taqman法检出GAPDH的下限为2．0×10^4拷贝，线性范围：2．0×10^4～2．0×10^10拷贝；SYBR GreenⅠ法检出GAPDH的下限为2．0×10^7拷贝，线性范围：2．0×10^7～2．0×10^10拷贝；普通PCR检出GAPDH的下限为2．0×10^6拷贝，线性范围：2．0×10^7～2．0×10^9拷贝。结论 Taqman荧光定量PCR法比SYBR GreenⅠ荧光定量PCR法和普通PCR法的灵敏度高，线性范围宽，是核酸水平对GAPDH定量的好方法。
Objective In order to find out the best method for GAPDH quantification, the levels of housekeeping gene GAPDH mRNA were detected by Taqman fluorescence quantitative polymerase chain reaction（FQ-PCR）, SYBR Green Ⅰ FQ-PCR and conventional PCR, respectively. Methods GAPDH plasmids were constructed by Blue-White Clone method and checked by PCR and EcoR Ⅰ. One of the correct clones was selected for isolating a large amount of plasmids. The selected plasmids were then 10-fold diluted and served as standard. Standard curves were prepared using the data from Taqman FQ-PCR, SYBR Green Ⅰ FQ- PCR and conventional PCR, respectively. The sensitivity and linear range of the three methods were then analyzed. Results The detectable thresholds for Taqman FQ-PCR, SYBR Green I FQ-PCR and conventional PCR were 2.0 × 10^4 , 2.0 ×10^7 and 2.0 × 10^6 DNA copies, respectively. The linear range for these three methods were 2.0×10^4 -2.0×10^10 copies, 2.0×10^7 -2.0× 10^10 copies and 2.0 ×10^7 - 2. 0 ×10^9 DNA copies, respectively. Conclusion Taqman FQ-PCR is a better method for quantifying housekeeping gene GAPDH mRNA than SYBR Green Ⅰ FQ-PCR and conventional PCR.
出处
《首都医科大学学报》
CAS
2006年第2期210-213,共4页
Journal of Capital Medical University
关键词
PCR
GAPDH
荧光定量
polymerase chain reaction
GAPDH
fluorescence quantification
