Chemical sequencing of restriction fragments 3'-end-labeled with [35S]dATPalphaS.
Although the phosphorous radioisotope 32P is routinely used in Maxam-Gilbert sequencing, it presents disadvantages that adversely affect safety and ease of use, resolution, and DNA degradation rates. Here, we introduce a new protocol utilizing the alternative isotope 35S for 3'-end-labeling DNA restriction fragments. In our method, plasmid DNA is labeled with [35S]dATPalphaS and 7 Sequenase Version 2.0. We have shown that bands on Maxam-Gilbert sequencing gels are sharp with extremely low background. In addition, a single labeling reaction produces DNA sufficient for 80 sequencing lanes, and the labeled DNA can be utilized for prolonged periods of time without significant degradation. We have further demonstrated the utility of our 35S-end-labeling procedure by successfully mapping the sequence-specificity of DNA damage induced by photoexcited riboflavin. Overall, we have shown that 35S can be used as a safe and practical alternative to 32P in the 3'-end-labeling of DNA restriction fragments.
Yang
Xia
X
Department of Chemistry, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303-3083, USA.
Grant
Kathryn B
KB
D001345:Autoradiography; D004259:DNA-Directed DNA Polymerase; D003838:Deoxyadenine Nucleotides; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013462:Sulfur Radioisotopes; D013873:Thionucleotides
D003838:Deoxyadenine Nucleotides; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; C015109:2'-deoxyadenosine 5'-O-(1-thiotriphosphate); C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase