Norris M D, Stewart B W
Children's Leukaemia and Cancer Research Unit, Prince of Wales Children's Hospital, Randwick, NSW, Australia.
FEBS Lett. 1988 Feb 15;228(2):223-7. doi: 10.1016/0014-5793(88)80003-6.
The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage luminal diameter X174 were hybridized to the intact 'plus' strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose. After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.
从苯甲酰化二乙氨基乙基纤维素上完全洗脱单链DNA所需的咖啡因浓度与多核苷酸长度成正比。使用一系列杂交分子研究了苯甲酰化二乙氨基乙基纤维素色谱法在分离和测定双链DNA中单链区域方面的应用。噬菌体腔直径X174复制形式的限制性片段与完整的“正”链杂交，从而形成在千碱基范围内具有单链和/或双链区域的杂交体。一系列这样的杂交制剂在苯甲酰化二乙氨基乙基纤维素上进行咖啡因浓度梯度洗脱。经对数转换后，洗脱咖啡因浓度与单链长度之间可呈现线性关系（R = 0.94），而与相关双链区域的长度或给定片段内未配对核苷酸的位置无关。因此，苯甲酰化二乙氨基乙基纤维素色谱法可用于在制备规模上分离和表征含有单链区域的双链DNA。
