低溫冷凍是否會破壞核酸分子？

01-24

如題。核酸分子會不會因為冷凍產生的小冰晶之類的被打斷或破壞什麼的？

看你怎麼凍。

液氮速凍正是因為可以避免冰晶的出現，直接凍成玻璃體，才能夠保存細胞活性。

即便如此，操作過程不可能都完美。

@孫岩你們實驗室的老師也一定說過反覆凍融對 DNA 活性的影響吧？

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實驗中，反覆凍融的確對DNA活性有影響，但一直機理不明。查到這個 http://onlinelibrary.wiley.com/doi/10.1111/j.1439-0531.2011.01815.x/abstract

裡面說

However, the direct mechanism through which intracellular ice formation could affect sperm DNA integrity is still unclear. There is strong evidence indicating that the freezing–thawing process induces oxidative stress that is associated with free radicalmediated damage to sperm nuclear DNA. Owing to the lack of functional DNA-repair mechanisms, it is possible that frozen-thawed spermatozoa are more vulnerable to oxidative attack. Evidence has been shown that the freezing process triggers apoptotic-like cascades in sperm cells, which activate caspases and subsequently specific endonucleases, resulting in sperm nuclear DNA fragmentation. However, the recent findings by Thomson et al. (2009) have demonstrated that DNA fragmentation in frozen-thawed human spermatozoa could occur independently of caspase and endonuclease activation and likely occur in apoptotic cells, suggesting that the apoptotic pathway could have a minor role in inducing DNA fragmentation in frozen-thawed spermatozoa. The findings of this study re-affirm that sperm DNA fragmentation incurred during freezing–thawing of human semen might be predominately attributed to oxidative stress rather than the activation of apoptotic-like cascades. Whether oxidative stress or apoptosis accounts for a significant proportion of DNA fragmentation in boar spermatozoa following freezing–thawing is still unclear. The main cause of sperm DNA damage in frozen-thawed semen is still open for discussion.

總結一下就是細胞內冰晶應該傷害沒那麼大，但是我們對DNA損傷修復機制了解太少，所以不知道是因為氧化，還是低溫啟動類似於細胞凋亡的過程。總之反覆凍融不好就是了。

首先，核酸分為大片段的和小片段的。先說小片段的，舉個例子，引物，我試過含引物的PCR反應液，反覆凍融30次以上，對比發現曲線完全一致，由此，小片段是耐凍融的。大片段以質粒為例，經過凍融以後，會出現擴增效率提高的現象，這是因為超螺旋結構受到破壞，發生線性化，更容易擴增了。由此，反覆凍融，會影響大片段核酸的穩定性。至於單純低溫，凍融一次，基本不會對核酸有太大影響。

看你凍什麼，怎麼凍。太多這樣沒條件限制的問題讓人怎麼回答

不會，我們實驗室提取的dna rna樣品都是在-80冰箱保存的，另外，如果低溫會破壞核酸分子的話，細胞在液氮里保藏也就沒有意義了。因為破壞掉了是肯定不能復甦的。dna在常溫下才不穩定容易降解使其結構受到破壞，rna在常溫下降解，並受到rna酶的影響分解掉。在低溫下酶活性會迅速降低從而保證核酸不被分解降解