Gaastra W
Department of Microbiology, The Technical University of Denmark, Lyngby, Denmark.
Methods Mol Biol. 1985;2:343-50. doi: 10.1385/0-89603-064-4:343.
As mentioned in Chapter 51 , DNA sequence analysis is based on high-resolution electrophoresis on denaturing polyacrylamide gels. In both the partial chemical cleavage method ( Chapter 51 ) and the partial resynthesis method ( Chapter 53 ), the labeled DNA fragments to be separated have one common end and the other end varies in length. The minimal size difference of DNA fragments in adjacent tracks of the gel is one nucleotide. The resolution of the gel should therefore be high enough to see two DNA molecules differing in size by one nucleotide as two separate bands. The resolution of the gel can be increased by decreasing the thickness of the gel and by decreasing the volume of the sample applied to each slot. Several percentages of polyacrylamide are routinely used for DNA sequence gels (i.e., 6, 8, 12, and 20% acrylamide) depending on which part of the sequence is to be read. The higher acrylamide percentage gels are used to determine the sequence of the first 50-100 nucleotides. Very long sequences can be read if the size of the gel is increased considerably, e.g., to 1 m. On these gels up to 400 nucleotides can be read. The gels are run under conditions that assure a temperature between 50-60°C at the surface of the plates. This, together with the high concentration of urea in the gel, is to make sure that the DNA does not renature during electrophoresis. The DNA molecules are denatured before loading and should not renature in order to obtain the correct distance between bands differing in size by only one nucleotide.
如第51章所述，DNA序列分析基于在变性聚丙烯酰胺凝胶上的高分辨率电泳。在部分化学切割法（第51章）和部分重新合成法（第53章）中，待分离的标记DNA片段都有一个共同的末端，而另一端长度不同。凝胶相邻泳道中DNA片段的最小尺寸差异为一个核苷酸。因此，凝胶的分辨率应足够高，以便将大小相差一个核苷酸的两个DNA分子视为两条分开的条带。可以通过减小凝胶厚度和减少加到每个槽中的样品体积来提高凝胶的分辨率。根据要读取序列的哪一部分，通常使用几种不同百分比的聚丙烯酰胺用于DNA序列凝胶（即6%、8%、12%和20%的丙烯酰胺）。较高百分比丙烯酰胺的凝胶用于确定前50 - 100个核苷酸的序列。如果凝胶尺寸大幅增加，例如增加到1米，就可以读取很长的序列。在这些凝胶上可以读取多达400个核苷酸。凝胶在确保平板表面温度在50 - 60°C之间的条件下运行。这与凝胶中高浓度的尿素一起，是为了确保DNA在电泳过程中不会复性。DNA分子在加样前变性，并且不应复性，以便获得大小仅相差一个核苷酸的条带之间的正确距离。
