Seryl-histidine as an alternative DNA nicking agent in nick translation yields superior DNA probes and hybridizations.
Nick translation is a commonly used method for labeling DNA to make DNA hybridization probes. In this approach, the use of DNase I to generate nicks in double-stranded DNA presents an inherent drawback, because the enzyme's high rate of reaction causes significant fragmentation and shortening of the hybridization probes. Based on our recent findings regarding the nucleolytic activity of the dipeptide seryl-histidine (Ser-His) and generation of free 3' hydroxyl and 5' phosphate groups at the cleavage sites of the substrate DNA by Ser-His, it was hypothesized that this disadvantage may be overcome by using Ser-His in place of DNase I as an alternative DNA nicking agent. In this study we demonstrate that like DNase I, Ser-His randomly nicks DNA, but the dipeptide has a much lower rate of reaction that enables more complete labeling of the DNA probes with less fragmentation. DNA probes labeled through nick translation using Ser-His as the DNA nicking agent were consistently larger in size and exhibited significantly higher specific activities, and enhanced hybridization signals in Southern blot analyses compared to control DNA probes that were made using DNase I as the nicking agent. Furthermore, the degree of nicking and consequently the quality of the probes could be easily controlled by adjusting the temperature and time of the Ser-His nicking reaction. These results affirm our hypothesis that Ser-His can serve as an alternative DNA nicking agent in nick translation to yield superior DNA probes and hybridization results and suggest the possible general utility of Ser-His for wide range of biological and biomedical applications that require more moderated nicking of nucleic acids. Based upon these and computer modeling results of Ser-His, a mechanism of action is proposed to explain how Ser-His may nick DNA.
Li
Yunsheng
Y
Edison Biotechnology Institute, 109 Konneker Research Center, Ohio University, the Ridges, Athens, OH 45701, USA.
Hatfield
Scott
S
Li
Jing
J
McMills
Mark
M
Zhao
Yufen
Y
Chen
Xiaozhuo
X
D001665:Binding Sites; D015139:Blotting, Southern; D003198:Computer Simulation; D004247:DNA / Q000032:analysis / Q000378:metabolism; D053938:DNA Fragmentation; D015342:DNA Probes / Q000032:analysis / Q000096:biosynthesis* / Q000592:standards; D003850:Deoxyribonuclease I / Q000378:metabolism / Q000592:standards; D004151:Dipeptides / Q000378:metabolism* / Q000592:standards; D020287:In Situ Nick-End Labeling / Q000379:methods* / Q000592:standards; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization / Q000379:methods*
D015342:DNA Probes; D004151:Dipeptides; C422849:seryl-histidine; D004247:DNA; D003850:Deoxyribonuclease I