Wood T G, Lingrel J B
J Biol Chem. 1977 Jan 25;252(2):457-63.
A complementary DNA (cDNA) copy of mouse globin mRNA was synthesized using the RNA-dependent DNA polymerase from avian myeloblastosis virus and the oligo(dT) covalently attached to cellulose as primer. All four deoxyribonucleotide triphosphates, NaCl, the globin mRNA template, and an oligo(dT) primer were required for optimal synthesis of cDNA. By saturating the primer sites using a 3-fold excess of mRNA, sufficient concentrations of immobilized cDNA could be synthesized to allow the hybridization reactions to be performed using an excess of globin cDNA. Conditions which permitted the annealing of globin mRNA to cDNA-cellulose were selected and the sequence specificity for hybridization to cDNA-cellulose was determined using 28 S ribosomal RNA, polyadenylic acid, and mouse L-cell RNA. Both analytical and preparative applications of this chromatographic medium were explored. When radioactively labeled poly(A)-containing 9 S RNA isolated from nucleated erythroid cells was analyzed by affinity chromatography on globin cDNA-cellulose, 46 per cent of the applied radioactivity hybridized to the cDNA-cellulose column. Only 1 per cent of the labeled RNA was retained by the column during reapplication of the unbound fraction, while 96 per cent of the bound RNA reannealed to cDNA-cellulose. Hybridizations utilizing unfractionated RNA extracts from either mouse reticulocytes or nucleated erythroid cells provided a one-step purification method for globin mRNA sequences. The relative purity of the RNA isolated by cDNA-cellulose affinity chromatography was determined by hybridization kinetic analysis. The cDNA-bound fraction obtained from the unfractionated RNA of either cell type was shown to have a Crt1/2 of 2.7 x 10-3. This represents a 60-fold purification of the globin sequences present in reticulocyte polysomal RNA and a 280-fold enrichment of the globin mRNA in nucleated erythroid cells. Hybridization to cDNA-cellulose did not result in any change in the sedimentation rate of globin mRNA. Furthermore, experiments were performed which demonstrated that the globin mRNA isolated by hybridization to cDNA-cellulose retained its biological activity when assayed in a wheat germ cell-free lysate.
使用来自禽成髓细胞瘤病毒的依赖RNA的DNA聚合酶和共价连接到纤维素上的寡聚（dT）作为引物，合成了小鼠珠蛋白mRNA的互补DNA（cDNA）拷贝。最佳合成cDNA需要所有四种脱氧核糖核苷三磷酸、氯化钠、珠蛋白mRNA模板和寡聚（dT）引物。通过使用3倍过量的mRNA饱和引物位点，可以合成足够浓度的固定化cDNA，以允许使用过量的珠蛋白cDNA进行杂交反应。选择了允许珠蛋白mRNA与cDNA-纤维素退火的条件，并使用28S核糖体RNA、聚腺苷酸和小鼠L细胞RNA确定了与cDNA-纤维素杂交的序列特异性。探索了这种色谱介质的分析和制备应用。当通过在珠蛋白cDNA-纤维素上进行亲和色谱分析从有核红细胞中分离的放射性标记的含聚（A）的9S RNA时，46%的施加放射性与cDNA-纤维素柱杂交。在未结合部分重新应用期间，只有1%的标记RNA被柱保留，而96%的结合RNA重新与cDNA-纤维素退火。利用从小鼠网织红细胞或有核红细胞中提取的未分级RNA进行杂交，为珠蛋白mRNA序列提供了一种一步纯化方法。通过杂交动力学分析确定了通过cDNA-纤维素亲和色谱分离的RNA的相对纯度。从两种细胞类型的未分级RNA中获得的与cDNA结合的部分显示其Crt1/2为2.7×10-3。这代表了网织红细胞多聚核糖体RNA中存在的珠蛋白序列的60倍纯化以及有核红细胞中珠蛋白mRNA的280倍富集。与cDNA-纤维素杂交不会导致珠蛋白mRNA沉降速率的任何变化。此外，进行的实验表明，通过与cDNA-纤维素杂交分离的珠蛋白mRNA在小麦胚芽无细胞裂解物中进行测定时保留了其生物活性。
