S_1核酸酶作用与微环DNA分子的克隆策略_cropped

合成探针 ,进行 Southern 杂交 。图 3b 显示 ,酶切的插入片段 被探针杂交 ,表明克隆的外源片段是 pC3 ,核酸序列测定其 长度为 537bp ,也与预期结果一致 。pC3 的序列已被 GenBank 4 , 将开环转变成线形 。这 说 明 经 过 S处切开另一条单链 1 () 收录 收录号 :AF522195。 核酸酶作用和电泳分离后形成锐带的线形分子 ,结构完整 , 末端平滑 ,回收后可用于克隆 。 生 物 工 程 学 报 19 卷 242 量重复序列 ,这提示 ,DNA 的二级结构以及可能存在的某些 未知修饰抑制了限制性内切酶的作用 ,使以限制性内切酶线 形化为基础的分子克隆无法实施 。对于难以用内切酶线形 化的双链微环 DNA 分子 ,尤其是像线粒体类质粒那样需经 纯化的少量样品 ,采用以 S核酸酶线形化为基础进行克隆 1 是省时而有效的途径 。 )(REFERENCES参考文献 Hoflack L , Seurinck J , Mahillon J . Nucleotide squence and caracter2 1 ] ization of the cryptic B acillus thuingiensis plasmid p GI3 reveal a new 图 2 S核酸酶对 pC3 的作用 1 Fig. 2 Digestion of pC3 DNA by Snuclease ( ) 1 family of rolling circle replicons. J B acteriol , 1997 , 179 6: 5000 Samples were analyzed by 2 . 5 % agarose gel electrophoresis. 1 and 3 . - 5008 Samples digested with 45u and 15u Snuclease , respectively ; 2 . sample 1 Gobbi E , Carpanelli A , Firrao G et al . The Cryphonenectria parasit2 2 ] not treated with any enzymes ; M. DNA marker . CCC , OC and L repre2 ica plasmid pUG1 contains a large ORF with motifs of family B DNA sents covalently closed circle , open circle and linear conformation , respec2 () polymerases. Nucleic Acids Res , 1997 , 25 16: 3275 - 3280 tively Bengtsson B O and Andersson H. The population genetics of plant 3 ] mitochondrial plasmids. J Theor Biol , 1997 , 188 : 163 - 176 Wiegand R C , Godson G N and Randding C M. Specificity of the S 4 ] 1 ( ) nuclease from Aspergillus oryzae . J Biol Chem , 1975 , 250 22 : 8848 - 8855 Levings III C S , Vasil I K. The Molecular biology of plant mitochon2 5 ] dria . Netherland : Kluwer Academic , 1995 , pp . 61 - 91 Goblet J P , Boutry M , Duc G et al . Mitochondrial plasmid2like mol2 6 ] ecules in fertile and male sterile Vicia faba L . Plant Moleluar Biolo2 gy , 1983 , 2 :305 - 309 Ausubel F M , Brent R , Kingston R E et al . Short Protocols in Mo2 7 ] rd lecular Biology. 3ed , John Weley & Sons , Inc , 1995 Kemble R J , Gunn R E , Flavell R B. Classification of normal and 8 ] male sterile cytoplasms in maize II. electrophoretic analysis of DNA species in mitochondria . Genet , 1980 , 95 :451 - 458 图 3 pC3 重组子鉴定 Fig. 3 Identification of recombinant pC3 DNA clone Wilson A J , Chourey P S. A rapid inexpensive method for the iola2 9 ] () ( ) Electrophoresis was done with 1 . 2 % agarose gel . Lane 1 in 3 a, 3 btion of restrictable mitochondrial DNA from various plant sources. ( ) and 3 b′are all loaded with the total DNA of cucumber mitochondrium. Plant Cell Reports , 1984 , 3 :237 - 239 Lane 2 : recombinant pC3 sample after digestion with EcoR ?and Pst ?. ( ) ( ) ( ) BAI Y L 白艳玲, YANG ZH L 杨之龙, YU G W于国武et 10 The arrow indicates the inserted fragment cut down by restriction enzymes. () al . Isolation and identification of plasmid2like DNAs in cucumber b′is the Southern hybridization result of b. M : DNA marker DL2000. CCC and OC represents covalently closed circle and open circle conforma2 mitochondria. Acta Scientiarum Naturaliun Universitatis Nankaiensis tion , respectively ( ) () 南开大学学报, 2002 , 35 2: 97 - 100 Sambrook J ,Fritcsh E F , Maniats T. Molecular cloning , A laboratory 11 根据测定的核酸序列 ,用已知的单切点限制酶消化类质 nd manual 2 ed ,1989 粒 pC3 ,仍未切开该分子 。序列分析表明 ,pC3 分子上存在大 The Action of SNuclea se and a Cloning Strategy f or Microcircular D NAs 1 3BAI Yan2Ling YANG Zhi2Long QIAO Ming2Qiang ZHANG Xiu2Ming ZHOU Jing GAO Cai2Chang ( )College of Life Sciences , Nankai University , Tianjin 300071 Received : 1120822002 () This work was supported by Grant from NSFCNo . 39770414. 3 Corresponding author : Tel : 86222223503692 ; Fax : 86222223508800 ; E2mail : gaocc @public . tpt . tj . cn ) (Abstract Snuclease from Aspergillus oryzaeis a specific enzyme to degrade single stranded DNA or RNA molecules. It has 1 been reported to be able to convert superhelical circular DNA molecules into open circle or linear forms under certain conditions , but this function has not been well explored. In order to use the action of Snuclease to linearize circular DNA and develop a novel way of cloning microcircular DNAs , 1 the pUC19 was used to investigate the relationship between the linearization efficiency of Snuclease and the amount of enzyme 1 used. By this way the optimal conditions for linearization of circular DNAs by Snuclease would be determined. 1 μ0 . 3u to 17u Snuclease per 100ng pUC19 DNA was added into a 25L system , respectively , to perform the reaction. The 1 effectiveness of enzyme digestion was realized by electrophoresis in a 1 . 2 % agarose gel . The results showed that along with the increase in enzyme amount from 0 . 3u to 17u a gradual decrease in the superhelical form , a gradual increase in the linear form and then in the circular form was obvious. The conversion from superhelical form to linear and circular form was directly related to the enzyme amount used. A higher proportion of linear DNA molecules was achieved by using 5 to 17u Snuclease per 100ng 1 DNA. Besides , electrophoretic mobility of the Snuclease2linearized pUC19 was the same as that of the linear form produced by 1 restriction enzyme digestion. According to the result of