DNA是无色的,所以我们想要看见它们,就要用到染色技术。自PCR技术成熟之后,DNA染色法被大量开发,我们想用最便宜、简单而且安全的方法进行最灵敏、有效的染色。小编今天就聊聊几种常见的染色剂。这些染色剂有的是荧光染料(Fluorescent dyes),有的是非荧光(Non-fluorescent dyes)。荧光和非荧光染料各有优劣,也不能说哪一种绝对的好,要根据情况而定。
1荧光染料溴化乙锭(Ethidium Bromide, EtBr)
EtBr分子结构
最常用、最便宜的DNA/RNA染色剂(结构如上图)。在琼脂糖胶里,EtBr的灵敏度是检测到至少1 ng 的DNA。染色过程可以是通过制作含有EtBr的琼脂糖胶,或者是在跑完胶后对整个胶体用EtBr稀释液染色。EtBr在紫外光照射下散发出橘黄色的光,当EtBr与DNA结合后,发光量大大增强。这就是为什么一整块经过EtBr处理的琼脂糖胶,在紫外光下,只有DNA所在的地方会发出强光,而不是整块胶都强光闪闪。EtBr即给DNA染了色,也标记了DNA,让我们了解到目标DNA的长短。唯一的不好就是,EtBr是潜在的可致癌诱变剂,所以在使用的时候一定要避免直接接触,使用有效防护,注意安全。
2 荧光染料SYBR系列
Thermo Fisher 出产的SYBR系列主要有三种:SYBR-Green,SYBR-Gold和SYBR-safe。SYBR-Gold有超高灵敏度(最低检测25 pg DNA),而SYBR-Green灵敏度稍低一些(大概在最低 60 pg DNA),SYBR-safe和EtBr灵敏度相似。不同于EtBr,SYBR系列染料不仅可以被紫外光激发,还可以被蓝光激发,因此避免了紫外光可能对DNA样本造成的破坏或突变。根据名字,我们也可以知道SYBR-safe是安全的、无害的,但另外两种却是潜在致癌物,使用时要小心。小编以前用过SYBR-safe染料,好用是好用,可是贵啊~~~
3荧光类其他染料
Biotium公司的Gel-Red和Gel-Green。两种染料的灵敏度和EtBr类似,Gel-Red只能被紫外光激发,Gel-Green只能被蓝光激发。这两种染料是完全无害的,没有致癌致畸的危害。小编也用过Gel-Red,效果不错,价钱比SYBR-safe稍微便宜一些,而且还是无毒无害的。性价比上还是不错的,但不管怎样肯定是不会比EtBr便宜的!
小编还听说过Lonza Bioscience 公司的GelStar 和Amresco公司的EZ-vision系列。只是从来没用过,也不知道效果如何。市面上还有很多荧光类的DNA染料,就不一一罗列了。
4非荧光类染料
非荧光染料的灵敏度肯定是大大不如荧光类染料的,但是非荧光类染料无需紫外光激发,能够减少对DNA的破坏。常见的非荧光染料有噻嗪类染料(Thiazine dyes),比如亚甲基蓝(Methylene blue)。除此以外,还有尼罗蓝类染料(Nile blue dyes,Nile blue A)。非荧光类染料都是与DNA进行离子键结合来显示DNA的位置的。
染料千千万,你们用什么呢,欢迎留言讨论!
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