Brown T D, Balmain A
Nucleic Acids Res. 1979 Dec 20;7(8):2357-68. doi: 10.1093/nar/7.8.2357.
The effect of different levels of mercury substitution on the rate and extent of hybridisation of globin mRNA with a complementary DNA (cDNA) copy has been investigated. It was found that mercuration significantly reduces both the rate of hybridisation and the extent of the reaction, but that these effects are abolished when at least stoichiometric amounts of 2-mercaptoethanol are included in the hybridisation medium. As a preliminary to using this technique to isolate specific groups of sequences after long-term hybridisations, we have investigated both the rate of demercuration of RNA and its retention on thiol-sepharose columns after extended incubation under commonly employed hybridisation conditions at 43 degrees or 60 degrees. Retention was essentially quantitative even after incubation times of 300 hours at 43 degrees, but decreased significantly after 48 hours at 60 degrees. It is concluded that thiol-sepharose chromatography offers considerable advantages over hydroxyapatite chromatography for the recovery of hybridised sequences, particularly with regard to the lower levels of non-specific binding obtained and its ability to distinguish directly between DNA-DNA and DNA-Hg RNA hybrids.
研究了不同汞取代水平对珠蛋白mRNA与互补DNA（cDNA）拷贝杂交速率和程度的影响。发现汞化显著降低了杂交速率和反应程度，但当杂交介质中至少包含化学计量的2-巯基乙醇时，这些影响就会消除。作为在长期杂交后使用该技术分离特定序列组的初步研究，我们研究了在43℃或60℃常用杂交条件下长时间孵育后RNA的脱汞速率及其在硫醇-琼脂糖柱上的保留情况。即使在43℃孵育300小时后，保留率基本也是定量的，但在60℃孵育48小时后显著下降。得出的结论是，对于杂交序列的回收，硫醇-琼脂糖色谱法比羟基磷灰石色谱法具有相当大的优势，特别是在获得较低水平的非特异性结合以及直接区分DNA-DNA和DNA-Hg RNA杂交体的能力方面。
