Restriction endonucleases digesting DNA in PCR buffer

编号 zgly0000390656

文献类型 期刊论文

文献题名 Restriction endonucleases digesting DNA in PCR buffer

作者 LIU Xue-dong  ZHENG Dong  ZHOU Yan-na  MAO Wei-wei  MA Jian-zhang 

作者单位 College of Wildlife Resources  College of Life Science  College of Wildlife Resources 

母体文献 林业研究: 英文版 

年卷期 2005,16(1)

页码 58-60,i003-i004

年份 2005 

分类号 Q55  Q783 

关键词 限制性内切酶  PCR缓冲液  酶切活性  RFLP  SSCP 

文摘内容 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。