Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA.
Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA.
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DOI：
10.1093/nar/gkm052
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发表时间：
2007
影响因子：
14.9
通讯作者：
Komiyama M
中科院分区：
文献类型：
--
作者：
Yamamoto Y;Mori M;Aiba Y;Tomita T;Chen W;Zhou JM;Uehara A;Ren Y;Kitamura Y;Komiyama M
伪互补肽核酸（pseudo-complementary peptide nucleic acid, pcPNA）末端附着一个单磷酸基团，两个修饰后的pcPNA与Ce(IV)/EDTA结合，进行双链DNA的位点选择性水解。这种pcPNAs的化学修饰明显加快了位点选择性DNA断裂。这些第二代人工限制性DNA切割器（ARCUTs）对目标序列的区分非常严格，即使只有一个DNA碱基对被改变为另一个碱基对也不会发生断裂。通过同时使用两个激活的ARCUTs， DNA底物在两个预定的位点被选择性地切割，所需的片段被剪切和克隆。即使目标位点被甲基转移酶甲基化，并且不受相应限制性内切酶的影响，ARCUT的DNA切割也是成功的。此外，在大肠杆菌基因组DNA（由4,600,000 bp组成）的靶位点选择性切割已经证实了ARCUT操作巨大DNA的潜力。所有这些结果表明，ARCUTs具有广泛的应用前景。
A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000 bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.
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影响因子：
3.3
作者：
KOMIYAMA, M;SHIIBA, T;KODAMA, T
通讯作者：
KODAMA, T
影响因子：
14.9
作者：
Eisenschmidt, K;Lanio, T;Pingoud, A
通讯作者：
Pingoud, A
DOI：
10.1073/pnas.96.21.11804
发表时间：
1999-10-12
影响因子：
11.1
作者：
Lohse, J;Dahl, O;Nielsen, PE
通讯作者：
Nielsen, PE
影响因子：
14.9
作者：
Yamamoto, Y;Uehara, A;Komiyama, M
通讯作者：
Komiyama, M
影响因子：
1.6
作者：
Igawa, T;Sumaoka, J;Komiyama, M
通讯作者：
Komiyama, M