Wulfert Michael, Tapprich Christoph, Gattermann Norbert
Department of Hematology, Oncology and Clinical Immunology, Heinrich-Heine-University, Düsseldorf, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Feb 2;831(1-2):236-47. doi: 10.1016/j.jchromb.2005.12.024. Epub 2006 Jan 10.
Denaturing high pressure liquid chromatography (dHPLC) is an efficient method for discovery of unknown mutations by heteroduplex analysis of PCR fragments. For comprehensive mutation scanning of the whole 16.569 bp human mitochondrial genome, we developed a set of 67 primer pairs defining overlapping PCR fragments that are well suited for heteroduplex analysis. The aim of our optimization efforts was to ensure that point mutations are detectable at every nucleotide position of each amplicon. Some GC-rich regions of mitochondrial DNA (mtDNA) were found to have unfavourable melting profiles in all possible amplicons, therefore requiring GC-clamps at the end of one or both oligonucleotide PCR primers. Following detection of a heteroduplex pattern by dHPLC, our primers can also be employed for DNA sequencing to identify the underlying mutation. In case of heteroplasmic mutations with a low proportion of mutant mtDNA, a fragment collector is useful to recover the heteroduplex peak, which contains mutant and wildtype DNA molecules in a 1:1 ratio.
变性高效液相色谱法（dHPLC）是一种通过对PCR片段进行异源双链分析来发现未知突变的有效方法。为了对全长16569bp的人类线粒体基因组进行全面的突变扫描，我们开发了一组67对引物，用于定义重叠的PCR片段，这些片段非常适合异源双链分析。我们优化工作的目的是确保在每个扩增子的每个核苷酸位置都能检测到点突变。发现线粒体DNA（mtDNA）的一些富含GC的区域在所有可能的扩增子中都具有不利的解链图谱，因此需要在一个或两个寡核苷酸PCR引物的末端添加GC夹。通过dHPLC检测到异源双链模式后，我们的引物也可用于DNA测序以鉴定潜在的突变。对于突变型mtDNA比例较低的异质性突变，片段收集器有助于回收异源双链峰，该峰以1:1的比例包含突变型和野生型DNA分子。
