Schott H, Schwarz M
J Chromatogr. 1978 Sep 21;157:197-215. doi: 10.1016/s0021-9673(00)92335-5.
The preparative-scale chemical degradation of DNA from herring sperm to mixtures of purine oligonucleotides or deoxyriboadenylic acids is described. The deoxyriboadenylic acid mixture was separated into six fractions according to increasing ionic charge by column chromatography on QAE-Sephadex A-25. Of the first fraction, 75% was deoxyriboadenosine monophosphate; of the second, 99% consisted of a mixture of p(dA)2 and (dA)2p, while of the third 93% was pdAp. The remaining fractions contained more or less complex mixtures of longer-chain oligonucleotides which could be further separated by subsequent re-chromatography on QAE-Sephadex at pH 9.6. By application of paper chromatography to the fractions obtained from column chromatography, the pure nucleotide phosphates pdAp, p(dA)2p, p(dA)3p, p(dA)4p and the mixtures of sequence isomers p(dA)2, (dA)2p; p(dA)3, (dA)3p; and p(dA)6, (dA)6p could be isolated preparatively. The nucleotide phosphates were converted to (dA)2, (dA)3 or (dA)4 and the mixtures of sequence isomers to (dA)2, (dA)3 or (dA)6 by treatment with alkaline phosphatase. By this means dephosphorylated, paper chromatographically pure oligodeoxyriboadenylic acid may be obtained in a preparative scale from the hydrolysates. The structures of the nucleotides thus isolated were deduced from the absorbtion characteristics, the RF values and the results of enzymatic degradation.
本文描述了将鲱鱼精DNA化学降解为嘌呤寡核苷酸或脱氧核糖腺苷酸混合物的制备规模方法。通过在QAE-葡聚糖A-25上进行柱色谱,根据离子电荷增加将脱氧核糖腺苷酸混合物分离为六个级分。第一级分中75%是脱氧核糖腺苷单磷酸;第二级分中99%由p(dA)2和(dA)2p的混合物组成,而第三级分中93%是pdAp。其余级分含有或多或少更复杂的长链寡核苷酸混合物,可通过随后在pH 9.6的QAE-葡聚糖上重新色谱进一步分离。通过对柱色谱得到的级分进行纸色谱,可制备性地分离出纯核苷酸磷酸酯pdAp、p(dA)2p、p(dA)3p、p(dA)4p以及序列异构体混合物p(dA)2、(dA)2p;p(dA)3、(dA)3p;和p(dA)6、(dA)6p。核苷酸磷酸酯经碱性磷酸酶处理转化为(dA)2、(dA)3或(dA)4,序列异构体混合物转化为(dA)2、(dA)3或(dA)6。通过这种方式进行去磷酸化,可从水解产物中制备规模地获得纸色谱纯的寡脱氧核糖腺苷酸。由此分离出的核苷酸结构由吸收特性、Rf值和酶促降解结果推导得出。