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Biochemical quantitation of PM2 phage DNA as a substrate for endonuclease assay
Yoo Jin Joo1, Hee-Ju Kim, Jae Yung Lee
1Laboratory of Biochemistry, School of Life Sciences & Biotechnology, and BioInstitute, Korea University, Seoul 136-701, Korea.
Abstract:
Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4 degrees C; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.
Insights
This study details optimal storage and production methods for bacteriophage PM2, ensuring high-yield DNA purification for sensitive endonuclease assays. Proper phage handling is crucial for reliable fmol-range DNA analysis.
Area of Science:
- Molecular Biology
- Virology
Background:
- Bacteriophage PM2 possesses a double-stranded DNA genome in a closed circular form.
- PM2 DNA is valuable for sensitive nick-circle endonuclease assays.
- Maintaining phage infectivity for DNA purification requires specific storage conditions.
Purpose of the Study:
- To establish optimal storage conditions for bacteriophage PM2 to preserve infectivity.
- To determine conditions for high-yield phage progeny production for DNA preparation.
- To report proper quantitation of radioactivity and purified DNA yield for fmol-range endonuclease assays.
Main Methods:
- Investigated short-term (4°C) and long-term (liquid nitrogen, 7.5% glycerol) phage storage.
- Optimized bacteriophage production using a multiplicity of infection (MOI) of 0.03 at host OD600 of 0.3-0.5.
- Quantified radioactivity and purified DNA yield considering genome size and concentrations.
Main Results:
- Short-term storage at 4°C and long-term storage under liquid nitrogen with glycerol maintain phage infectivity.
- Optimal mass production of PM2 phage achieved at MOI 0.03, yielding approximately 214 phage particles per host.
- Established protocols for accurate radioactivity quantitation and DNA yield for fmol-range assays.
Conclusions:
- Specific storage protocols are essential for maintaining bacteriophage PM2 infectivity.
- Optimized infection parameters enable efficient mass production of PM2 phage.
- Accurate DNA quantitation and radioactivity measurements are critical for sensitive endonuclease activity detection.
