Green Michael R, Sambrook Joseph
Cold Spring Harb Protoc. 2021 Jul 1;2021(7):2021/7/pdb.prot100487. doi: 10.1101/pdb.prot100487.
In Southern blotting, DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through a standard agarose gel. The DNA is then denatured in situ and transferred from the gel to a solid support (usually a nylon or nitrocellulose membrane). The relative positions of the DNA fragments are preserved during their transfer to the membrane. The DNA is then fixed to the membrane and prepared for hybridization. Alternatively, DNA can be simultaneously transferred from the top and bottom surfaces of a single agarose gel to two membranes. This procedure is useful when the need arises to analyze the same set of restriction fragments with two different probes. Transfer of DNA fragments is rapid, but the efficiency is low because the agarose gel quickly becomes dehydrated as fluid is withdrawn from both sides. The method therefore works best when the target sequences are present in high concentration (e.g., when analyzing cloned DNAs [plasmids, bacteriophages, cosmids, PACs, or BACs] or less complex genomes [those of or ]). Too little mammalian genomic DNA is transferred to allow signals from single-copy sequences to be detected in a reproducible or timely fashion.
在Southern印迹法中，用一种或多种限制性内切酶消化DNA，然后通过标准琼脂糖凝胶电泳根据大小分离产生的片段。接着DNA在原位变性，并从凝胶转移到固相支持物（通常是尼龙或硝酸纤维素膜）上。DNA片段在转移到膜的过程中其相对位置得以保留。然后将DNA固定在膜上并准备进行杂交。另外，DNA可以从单个琼脂糖凝胶的顶面和底面同时转移到两张膜上。当需要用两种不同的探针分析同一组限制性片段时，此方法很有用。DNA片段的转移速度很快，但效率较低，因为随着液体从两侧抽出，琼脂糖凝胶会迅速脱水。因此，当靶序列浓度较高时（例如，分析克隆的DNA [质粒、噬菌体、黏粒、P1人工染色体或细菌人工染色体] 或不太复杂的基因组 [如 或 的基因组]），该方法效果最佳。转移的哺乳动物基因组DNA太少，无法以可重复或及时的方式检测到单拷贝序列的信号。
