Collins M, Myers R M
Genetics Institute, Cambridge, MA 02140.
J Mol Biol. 1987 Dec 20;198(4):737-44. doi: 10.1016/0022-2836(87)90214-2.
DNA molecules that differ by a single base-pair can be separated by denaturing gradient gel electrophoresis due to the sequence-specific melting properties of DNA. Base modifications such as methylation are also known to affect the melting temperature of DNA. We examined the final position of DNA fragments containing either 5-methyl-cytosine or 6-methyl-adenine in denaturing gradient gels. The presence of a single methylated base within an early melting domain resulted in a well-resolved shift in fragment position relative to the unmethylated sequence. In addition, fragments containing hemimethylated and fully methylated sites could be distinguished, and a proportionally larger shift was observed with an increasing number of methylated bases. Denaturing gradient gel electrophoresis thus provides a sensitive method for analyzing the methylation state of DNA, which is not dependent on the presence of restriction enzyme cleavage sites. We also demonstrate that denaturing gradient gel electrophoresis can be used to obtain a quantitative estimate of the change in helix stability caused by modification of one or two bases in a complex DNA sequence. Such estimates should allow more accurate modeling of melting of natural DNA sequences.
由于DNA的序列特异性解链特性，相差单个碱基对的DNA分子可通过变性梯度凝胶电泳分离。已知诸如甲基化等碱基修饰也会影响DNA的解链温度。我们研究了在变性梯度凝胶中含有5-甲基胞嘧啶或6-甲基腺嘌呤的DNA片段的最终位置。在早期解链区域内单个甲基化碱基的存在导致片段位置相对于未甲基化序列出现明显的迁移。此外，含有半甲基化和完全甲基化位点的片段可以区分，并且随着甲基化碱基数量的增加，观察到的迁移比例更大。因此，变性梯度凝胶电泳提供了一种分析DNA甲基化状态的灵敏方法，该方法不依赖于限制性酶切位点的存在。我们还证明，变性梯度凝胶电泳可用于定量估计复杂DNA序列中一个或两个碱基修饰引起的螺旋稳定性变化。这样的估计应该能更准确地模拟天然DNA序列的解链情况。
