New preparation of PM2 phage DNA and an endonuclease assay for a single-strand break.
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.
Jung
Sang-Oun
SO
Lab. of Biochemistry, Graduate School of Biotechnology, BioInstitute, Korea University, 136-701 Seoul, Korea.
Auh
Chung-Kyoon
CK
Ryu
Jae-Chun
JC
Kim
Joon
J
D001681:Biological Assay; D019240:Corticoviridae / Q000235:genetics*; D004249:DNA Damage; D004270:DNA, Circular / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects; D004279:DNA, Viral / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects; D004720:Endonucleases / Q000378:metabolism*; D011092:Polyethylene Glycols; D031161:Pseudoalteromonas / Q000821:virology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D012997:Solvents; D014466:Ultraviolet Rays
D004270:DNA, Circular; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D012997:Solvents; C044152:ribosomal protein S3; D011092:Polyethylene Glycols; D004720:Endonucleases