摘要
基于SYBR Green Ⅰ荧光染料与双链DNA（dsDNA）结合产生荧光的原理,建立一种高精度、高通量的双链DNA定量方法。将梯度稀释后的基因组DNA及已知浓度的λDNA与等体积的SYBR Green Ⅰ（4×）充分混合后,利用荧光定量PCR仪采集荧光信号,以ROX（1×）作为校正染料进行定量分析;同时利用紫外分光光度计对样品进行平行测定,比较该方法与紫外分光光度法的检测限与准确度。紫外分光光度法的检测限为2ng/μl,而SYBR Green Ⅰ荧光定量法的检测限可达到0.015ng/μl,并且在0.015-2ng/μl范围内,SYBR Green Ⅰ荧光强度与λDNA浓度呈线性关系（R2=0.9999）,比紫外分光光度法灵敏100倍以上,并可准确定量低纯度的DNA样品。此方法具有重复性好、高通量的特点,仅需少量的生物样本即可满足定量要求,为分子生物学研究及临床检验等多个领域提供了一种可靠的dsDNA定量方法。
An accurate and high-throughput fluorescence based method was developed for quantification of double strand DNA （dsDNA） using SYBR GreenⅠ dsDNA-binding dye. A series of diluted genomic DNA and λ.DNA were mixed with SYBR GreenⅠ （4 × ）, and the fluorescence signal was measured with real-time PCR instrument and calibrated with ROX （ 1 ×） as reference dye. The performance characteristics of the method were compared with spectrophotometric quantification based on ultraviolet absorption. The new dsDNA quantification using SYBR GreenⅠ exhibited linearity over a range of λDNA concentrations from 0. 015 to 2 ng/μl and was about 100-times more sensitive and accurate than spectrophotometric quantification. The new dsDNA quantification allowed DNA sample may contain impurities and only required a small quantity of tissue sample. It could be widely used in molecular biological and diagnostic applications as an accurate and high-throughput assay tool.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2008年第1期55-60,共6页
China Biotechnology
