Danielson Phillip B, Shelton Robert J, LaBerge Greggory S
University of Denver, Department of Biological Sciences, 2101 E. Wesley Avenue Rm. 211, Denver, Colorado 80208, USA.
Croat Med J. 2003 Aug;44(4):447-54.
To develop and evaluate heteroduplex forming templates (HFTs) as a common set of molecular standards for genotyping by denaturing high-performance liquid chromatography (DHPLC) using hypervariable regions of human mitochondrial DNA (mtDNA) as a model system.
Hypervariable regions 1 and 2 from the mtDNA D-loop of 22 maternally related and unrelated human volunteers were amplified by polymerase chain reaction (PCR) and individually mixed with each of three HFTs. Following denaturation and reannealing of the mixture, the resulting hetero- and homoduplicies were separated by DHPLC using temperature-modulated heteroduplex analysis.
Each of three HFTs, when cross-hybridized with a target mtDNA amplicon, induced the formation of an assemblage hetero- and homoduplex peaks, which were uniquely characteristic of a given mtDNA sequence variant. The mtDNA DHPLC profiles obtained in the current study were identical between maternal relatives and different between unrelated individuals--consistent with uniparental maternal inheritance of mtDNA in humans.
DHPLC in combination with a common set of HFTs targeted to a locus of interest can be used as a reliable means of genotyping. DHPLC profiles can be readily stored as a bit-coded string of hetero- and homoduplex peak retention times to form a searchable database. This approach to DHPLC genotyping will have immediate utility in extended pedigree analyses, where it will allow rapid sorting and/or confirmation of maternal lineages. Additional applications of DHPLC profiling include the discovery and scoring of clinically relevant nuclear and mitochondrial loci.
开发并评估异源双链形成模板（HFTs），将其作为一组通用的分子标准，以人线粒体DNA（mtDNA）高变区为模型系统，通过变性高效液相色谱（DHPLC）进行基因分型。
对22名母系相关和不相关的人类志愿者mtDNA D环的高变区1和2进行聚合酶链反应（PCR）扩增，并分别与三种HFTs中的每一种混合。混合物变性和重退火后，使用温度调制异源双链分析通过DHPLC分离产生的异源双链和同源双链。
三种HFTs中的每一种与目标mtDNA扩增子交叉杂交时，都会诱导形成一组异源双链和同源双链峰，这是给定mtDNA序列变异的独特特征。本研究中获得的mtDNA DHPLC图谱在母系亲属之间相同，在不相关个体之间不同——这与人类mtDNA的单亲母系遗传一致。
DHPLC结合针对感兴趣位点的一组通用HFTs可作为一种可靠的基因分型方法。DHPLC图谱可以很容易地存储为异源双链和同源双链峰保留时间的位编码字符串，以形成一个可搜索的数据库。这种DHPLC基因分型方法在扩展家系分析中将具有直接用途，它将允许快速分类和/或确认母系谱系。DHPLC分析的其他应用包括发现和评分临床相关的核和线粒体位点。
