Lim Kok Seong, Naviaux Robert K, Haas Richard H
Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA 92093-0935, USA.
Clin Chem. 2007 Jun;53(6):1046-52. doi: 10.1373/clinchem.2006.083303. Epub 2007 Apr 19.
In recent years, denaturing HPLC (DHPLC) has been widely used to screen the whole mitochondrial genome or specific regions of the genome for DNA mutations. The quantification and mathematical modeling of DHPLC results is, however, underexplored.
We generated site-directed mutants containing some common mutations in the mitochondrial DNA (mtDNA) tRNA(leu) region with different mutation loads and used PCR to amplify the gene segment of interest in these mutants. We then performed restriction digestion followed by slow reannealing to induce heteroduplex formation and analyzed the samples by use of DHPLC.
We observed a quadratic relationship between the heteroduplex peak areas and mutant loads, consistent with the kinetics of heteroduplex formation reported by others. This was modeled mathematically and used to quantify mtDNA mutation load. The method was able to detect a mutation present in a concentration as low as 1% and gave reproducible measurements of the mutations in the range of 2.5%-97.5%.
The quantitative DHPLC assay is well suited for simultaneous detection and quantification of DNA mutations.
近年来，变性高效液相色谱法（DHPLC）已被广泛用于筛选整个线粒体基因组或基因组的特定区域以寻找DNA突变。然而，DHPLC结果的定量和数学建模尚未得到充分探索。
我们构建了含有线粒体DNA（mtDNA）tRNA（leu）区域中一些常见突变且具有不同突变负荷的定点突变体，并使用聚合酶链反应（PCR）扩增这些突变体中感兴趣的基因片段。然后我们进行限制性酶切，随后缓慢复性以诱导异源双链体形成，并使用DHPLC分析样品。
我们观察到异源双链体峰面积与突变负荷之间呈二次关系，这与其他人报道的异源双链体形成动力学一致。我们对其进行了数学建模并用于量化mtDNA突变负荷。该方法能够检测到低至1%浓度的突变，并且在2.5%-97.5%的范围内对突变进行了可重复的测量。
定量DHPLC分析非常适合同时检测和定量DNA突变。
