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目的基于变性高效液相色谱技术,建立一种不需测序和杂交的新的mtDNA控制区多态性分析系统。方法mtDNA控制区序列(包括HVⅠ,HVⅡ和HVⅢ)被分为4个扩增片段,采用配对分析突变检测模式进行DHPLC分析。对DHPLC检测条件(包括柱温和洗脱梯度等)进行优化。对100个不同类型差异序列的组合配对以检验该方法的检测效力。结果10组序列相同的样本配对DHPLC图谱均只显示1个样品峰。对序列相差1个碱基～7个碱基、插入(/缺失)1个碱基、插入(/缺失)2个碱基等类型的90个扩增片段组合,用DHPLC进行分析,均得到≥2个样本峰的DHPLC图谱,序列差异检出率达100%。该技术可检测的异质性DNA成分的最小百分含量为10%。结论DHPLC-mtDNA控制区多态性分析系统快速、经济和实用,在检测mtDNA异质性方面较直接测序更灵敏。
Objective To establish a new mtDNA control region polymorphism analysis system without sequencing and hybridization based on denaturing high performance liquid chromatography (HPLC). Methods The sequences of mtDNA control region (including HVⅠ, HVⅡ and HVⅢ) were divided into four amplified fragments. DHPLC analysis was performed by paired-analysis mutation detection mode. DHPLC detection conditions (including column temperature and elution gradient, etc.) are optimized. The combination of 100 different types of differential sequences was paired to test the detection efficacy of this method. Results The 10 pairs of DHPLC samples with the same sequence showed only one sample peak. A sequence of 1 base to 7 base pairs, insert (/ deletion) of 1 base, insert (/ deletion) of 2 bases and other types of 90 amplified fragment combinations were analyzed by DHPLC, were ≥ DHPLC pattern of two sample peaks, the sequence difference detection rate of 100%. The minimum percentage detectable for heterogeneous DNA components is 10%. Conclusion The DHPLC-mtDNA control region polymorphism analysis system is rapid, economical and practical. It is more sensitive than direct sequencing in detecting mtDNA heterogeneity.