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荧光Cy5DNA扩增及酶切产物分离–检测
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Abstract:
dNTPs加入一定比例Cy5-dATP可以扩增荧光Cy5DNA，用于T5外切酶(T5exo)类DNA酶检测，关键是酶切产物分离–检测。[方法]本研究以1/1000 Cy5-dATP的dNTPs扩增5851 bp荧光Cy5DNA pET28a-xyn。基于纯化柱吸附DNA原理，首先确定2倍P3缓冲液(2×P3)为纯化柱吸附DNA最小用量，进而用纯化柱以2×P3分离Cy5DNA溶液、等量Cy5DNA经T5exo酶切反应液(Cy5DNA-T5exo)，分别得到洗脱液(含有Cy5DNA)和滤出液(含有酶切产物Cy5-dATP)。[结果]酶标仪定量检测显示，含120 ng Cy5DNA洗脱液荧光值5824、滤出液0。含120 ng Cy5DNA-T5exo滤出液荧光值7573、洗脱液0。激光共聚焦显微镜定性检测显示，Cy5DNA洗脱液有荧光、滤出液无。Cy5DNA-T5exo滤出液有荧光、洗脱液无。定量和定性检测结果与纯化柱吸附DNA、滤出酶切产物原理和功能相同。进而分析了Cy5DNA浓度、Cy5-dATP浓度与荧光值的定量关系。[结论]荧光Cy5DNA成功扩增，其酶切产物经纯化柱以2×P3成功分离，为荧光DNA扩增和用于DNA酶活性检测奠定了基础。
[Objective] Amplified with dNTPs added with a specific ratio of fluorescent Cy5-dATP, fluorescent Cy5DNA can assay exonuclease such as T5 DNA exonuclease (T5exo), key is separation-detection of digestion product. [Method] Amplified with dNTPs added with 1/1000 Cy5-dATP, a 5851 bp fluorescent Cy5DNA pET28a-xyn served for T5exo digestion. Basing on DNA-absorbing principle of DNA-clean column, a 2-flod buffer P3 (2×P3) that of DNA solution was determined as the least quantity of a column to absorb DNA. Next, a DNA-clean column with 2×P3 served to separate a solution containing Cy5DNAs or equal amount of Cy5DNAs digested by T5exo to collect filtrate and elute, respectively.[Result] Assayed by a microreader SpectraMax？ i3x, the filtrate and elute from 120 ng Cy5DNAs-T5exo solution had a fluorescence value of 7573 and 0, respectively. The elute and filtrate from 120 ng Cy5DNA solution had a fluorescence value of 5824 and 0, respectively. Assayed by a con-focal microscope, the filtrate from Cy5DNA-T5exo solution exhibited Cy5-dATP fluorescence, while the elute did not. The elute from Cy5DNA solution exhibited Cy5 fluorescence, while the filtrate did not. Both the quantity and the quality assay were in agreement with the principle and usage of DNA-clean column. Additionally, correlation was analyzed for concentration of Cy5DNA and Cy5-dATP with fluorescence intensity. [Conclusion] Cy5DNA pET28a-xyn was amplified successfully, and its T5exo digestion product Cy5-dATP was separated by a DNA-clean column with a 2×P3 buffer. The study provided a basis for fluorescent DNA amplification and usage in assaying DNA-cutting enzyme.
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