Longacre S S, Mach B
J Biol Chem. 1978 Oct 25;253(20):7500-7.
Recombinant plasmid DNA has been used to purify complementary cDNA by hybridization using a modification of sulfhydryl-Sepharose chromatography described by Dale and Ward ((1975) Biochemistry 14, 2458). Plasmid DNA containing cloned mouse globin or immunoglobulin sequences was mercurated and hybridized in solution to unpurified cDNA. The resulting hybrids were passed over a sulfhydryl-Sepharose column where mercurated polynucleotides are retained. After washing, cDNA hybridized to the mercurated plasmid DNA was melted in situ and eluted while the mercurated plasmid DNA remained bound to the column. The conditions for purification of DNA and RNA sequences are described. The purity of the cDNAs obtained by this method is analyzed by polyacrylamide gel electrophoresis and by hybridization. In addition, this nucleic acid purification procedure has been applied to two problems of general interest: (i) the sensitive titration of specific genes by saturation hybridization; (ii) the purification of DNA fragments bearing specific sequences from restriction endonuclease digests of total cellular DNA. The procedure is generally applicable to the purification by hybridization of any DNA or RNA sequence complementary to an available probe.
重组质粒DNA已被用于通过杂交来纯化互补cDNA，该杂交方法采用了戴尔和沃德（（1975年）《生物化学》14卷，2458页）描述的巯基-琼脂糖凝胶色谱法的一种改进方法。含有克隆的小鼠珠蛋白或免疫球蛋白序列的质粒DNA经汞化处理后，在溶液中与未纯化的cDNA杂交。所得杂交体通过巯基-琼脂糖凝胶柱，汞化的多核苷酸会被保留在该柱上。洗涤后，与汞化质粒DNA杂交的cDNA在原位解链并洗脱，而汞化质粒DNA仍与柱结合。文中描述了DNA和RNA序列的纯化条件。通过聚丙烯酰胺凝胶电泳和杂交分析了用该方法获得的cDNA的纯度。此外，这种核酸纯化方法已应用于两个普遍感兴趣的问题：（i）通过饱和杂交对特定基因进行灵敏滴定；（ii）从总细胞DNA的限制性内切酶消化物中纯化带有特定序列的DNA片段。该方法通常适用于通过杂交纯化与可用探针互补的任何DNA或RNA序列。
