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A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
New preparation of PM2 phage DNA and an endonuclease assay for a single-strand break
Sang-Oun Jung1, Chung-Kyoon Auh, Jae-Chun Ryu
1Lab. of Biochemistry, Graduate School of Biotechnology, BioInstitute, Korea University, 136-701 Seoul, Korea.
Abstract:
PM2 is a bacteriophage which has closed circular double-stranded DNA as a genome, which is the sole source for endonuclease assay for a single strand break in the fmol range. Therefore, it is important to isolate PM2 DNA with low control nicks for the endonuclease assay. Usually, the isolation method of phage DNA is to use ultracentrifugation which takes at least 4 days. In this report, a fast and effective method which takes only 2 days was developed to purify DNA using polyethylene glycol (PEG) 8000 and the yields of phage DNA isolated by these two methods were compared. The method using PEG 8000 increased the yield of PM2 DNA from 31.2% to 45.2%, and decreased the nick from 17.1% to 13.1%. Recently, the complete PM2 DNA genome sequence of 10,079 bp was published. The exact number of nucleotides of PM2 DNA is important for the correct enzyme assay which measures nicks generated by an endonuclease. The correct calculation of endonuclease activity of rpS3 for nick-circle assay was performed to measure single-strand breaks in this report.
Insights
A new method using polyethylene glycol (PEG) 8000 rapidly purifies bacteriophage (PM2) DNA. This PEG 8000 method improves DNA yield and reduces nicks, crucial for accurate endonuclease assays.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophage PM2 possesses a closed circular double-stranded DNA genome.
- PM2 DNA is essential for fmol-range single-strand break endonuclease assays.
- Accurate endonuclease assays require PM2 DNA with minimal nicks.
Purpose of the Study:
- To develop a faster and more effective method for purifying PM2 DNA.
- To compare the yield and nick levels of PM2 DNA isolated by ultracentrifugation versus a new PEG 8000 method.
- To ensure accurate endonuclease activity measurements for rpS3.
Main Methods:
- Developed a 2-day DNA purification protocol using polyethylene glycol (PEG) 8000.
- Compared DNA yields and nick percentages between the PEG 8000 method and traditional ultracentrifugation.
- Performed nick-circle assays to measure single-strand breaks using purified PM2 DNA.
Main Results:
- The PEG 8000 method increased PM2 DNA yield from 31.2% to 45.2%.
- The PEG 8000 method decreased DNA nicks from 17.1% to 13.1%.
- Accurate endonuclease activity calculation for rpS3 was achieved with the improved DNA preparation.
Conclusions:
- The PEG 8000 method offers a significantly faster and more efficient alternative for PM2 DNA purification.
- Reduced nick levels in PEG 8000 purified DNA enhance the reliability of endonuclease assays.
- This optimized DNA isolation is critical for precise measurement of single-strand breaks and enzyme activity.
