Abstract
Six commonly used restriction endonucleases (REs) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for additional magnesium supplemented as activator, REs, except EcoR I appeared star activity, completely digested unmethylated lambda DNA after overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all REs tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed directly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L−1 to 10 mmol·L−1 did not significantly affect activity of REs in PCR buffer. This simplified method for RE digestion of PCR products could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymorphism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.
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Foundation item: This research was supported by the Fund for ICP Cup of the Northeast Forestry University and partially by the Key Project (NO. 96-20) of State Forestry Administration.
Biography: LIU Xue-dong (1970-), female, Ph. D, Lecturer of Genetics in College of Wildlife Resources, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China.
Responsible editor: Chai Ruihai
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Xue-dong, L., Dong, Z., Yan-na, Z. et al. Restriction endonucleases digesting DNA in PCR buffer. Journal of Forestry Research 16, 58–60 (2005). https://doi.org/10.1007/BF02856857
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DOI: https://doi.org/10.1007/BF02856857