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shileinjut至尊木虫 (职业作家)
猪猪家族的猪拽拽
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[求助]
限制内切酶中Nick和Cleave两个术语的区别？
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| 如题“限制内切酶中Nick和Cleave两个术语的区别？”，看到文献中说这两个单词的含义是不一样的，不知道在切割DNA上，到底有啥区别？请各位大虾不吝赐教！ |
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- 有关双链DNA用nick酶切割后在用连接酶连接的问题
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3楼2012-10-23 20:48:33
tupac225
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小丹木木: 金币+2, 鼓励应助 2012-10-30 15:59:20
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小丹木木: 金币+2, 鼓励应助 2012-10-30 15:59:20
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至尊木虫呀，回答你的问题不胜惶恐呀， 你自己翻译吧 Nick translation [1] (or Head Translation) was developed in 1977 by Rigby and Paul Berg. It is a tagging technique in molecular biology in which DNA Polymerase I is used to replace some of the nucleotides of a DNA sequence with their labeled analogues, creating a tagged DNA sequence which can be used as a probe in Fluorescent in situ hybridization or blotting techniques. It can also be used for radiolabeling. [2] This process is called nick translation because the DNA to be processed is treated with DNase to produce single-stranded "nicks." This is followed by replacement in nicked sites by DNA polymerase I, which elongates the 3' hydroxyl terminus, removing nucleotides by 5'-3' exonuclease activity, replacing them with dNTPs. To radioactively label a DNA fragment for use as a probe in blotting procedures, one of the incorporated nucleotides provided in the reaction is radiolabeled in the alpha phosphate position. Similarly, a fluorophore can be attached instead for fluorescent labelling, or an antigen for immunodetection. When DNA polymerase I eventually detaches from the DNA, it leaves another nick in the phosphate backbone. The nick has "translated" some distance depending on the processivity of the polymerase. This nick could be sealed by DNA ligase, or its 3' hydroxyl group could serve as the template for further DNA polymerase I activity. Proprietary enzyme mixes are available commercially to perform all steps in the procedure in a single incubation. Nick translation could cause double-stranded DNA breaks, if DNA polymerase I encounters another nick on the opposite strand, resulting in two shorter fragments. This does not influence the performance of the labelled probe in in situ hybridization. [edit] References 1.^ Rigby PW, Dieckmann M, Rhodes C, Berg P (June 1977). "Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I". J. Mol. Biol. 113 (1): 237–51. PMID 881736. 2.^ Mathew CG (1985). "Radiolabeling of DNA by nick translation". Methods Mol. Biol. 2: 257–61. doi:10.1385/0-89603-064-4:257. PMID 21374202. |
2楼2012-10-23 16:24:44
