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shileinjut至尊木蟲 (職業(yè)作家)
豬豬家族的豬拽拽
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[求助]
限制內(nèi)切酶中Nick和Cleave兩個(gè)術(shù)語的區(qū)別？
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| 如題“限制內(nèi)切酶中Nick和Cleave兩個(gè)術(shù)語的區(qū)別？”，看到文獻(xiàn)中說這兩個(gè)單詞的含義是不一樣的，不知道在切割DNA上，到底有啥區(qū)別？請(qǐng)各位大蝦不吝賜教！ |
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- 有關(guān)雙鏈DNA用nick酶切割后在用連接酶連接的問題
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tupac225
金蟲 (正式寫手)
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小丹木木: 金幣+2, 鼓勵(lì)應(yīng)助 2012-10-30 15:59:20
感謝參與，應(yīng)助指數(shù) +1
小丹木木: 金幣+2, 鼓勵(lì)應(yīng)助 2012-10-30 15:59:20
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至尊木蟲呀，回答你的問題不勝惶恐呀， 你自己翻譯吧 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樓2012-10-23 16:24:44
酶切專家
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感謝參與，應(yīng)助指數(shù) +1
小丹木木: 金幣+3, 鼓勵(lì)應(yīng)助 2012-10-30 15:59:06
shileinjut: 金幣+30, ★★★很有幫助, 10 2012-12-08 15:44:38
感謝參與，應(yīng)助指數(shù) +1
小丹木木: 金幣+3, 鼓勵(lì)應(yīng)助 2012-10-30 15:59:06
shileinjut: 金幣+30, ★★★很有幫助, 10 2012-12-08 15:44:38
| Nick是指將雙鏈DNA中的一條年切開，而另一條保持完整（中文通常翻譯為切刻），cleave則是指將DNA的雙鏈都切開，當(dāng)然兩條鏈上的切割位點(diǎn)不一定相同，可以參見不同限制性內(nèi)切酶的說明書。 |
3樓2012-10-23 20:48:33
once1015
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4樓2012-10-30 12:27:36
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