Amplification of Fluorescent Cy5DNA and Separation-Detection of Digestion Product

Abstract

[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 so-lution 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 as-saying DNA-cutting enzyme.