通过对沙枣叶子HPLC色谱条件的选择和优化,确定了沙枣叶HPLC分析条件:流动相比例为乙腈∶水(7∶93v/v);流速:0.3ml/min;检测波长240nm;柱温:35℃;进样量为10ul;分析时间为80min。对主要色谱峰的保留时间和峰面积等色谱参数进行分析,结果表明各相关指标符合HPLC指纹图谱的评价标准。为沙枣叶子的指纹图谱研究提供了参考,进一步为沙枣的种质资源合理开发利用提供理论依据。该法操作简单,重复性好,可行性高。

Objective: Previous studies proved that somatic mutations of mitochondrial DNA(mtDNA) are strongly associated with tumorigenesis and tumor progression.However,conventional sequencing methods are not able to detect mtDNA mutations with high-throughput and high accuracy.In this study,we established a new approach for detecting mutations in mtDNA based on next-generation sequencing technology.Methods: mtDNA were amplified by PCR using genome DNA samples obtained from tumor tissue,paired normal tissue or peripheral blood.The PCR products went through blunt end ligation,sticky end ligation,or amino-modification before sequencing library preparation.The mtDNA libraries were sequenced utilizing Illumina HiSeq 2000 platform.The sequences were then compared with human mtDNA referencing sequences,and further analysis using bioinformatical methods.Results: After assessing the genomic DNA samples with different qualities,we found that using three pairs of PCR primers is applicable to most mtDNA enrichment reactions.We also found the amino-modified PCR primers could significantly improve the coverage uniformity,and reduce the sequencing costs.Conclusion: A method to optimize mtDNA enching and the coverage uniformity has been built up in this study based on next-generation sequencing technology.Further a sensitive mtDNA mutation detecting system with high specificity and high-throughput was aslo established.This system may provide a new method for research in mtDNA mutation detection and disease association studies.

通过对沙枣果实色谱条件的选择和优化,建立了沙枣果实的分析条件:色谱柱为Diamonsil C18(250×4.6mm,5μm);流动相为乙腈-水(5:95v/v);检测波长240nm;柱温:35℃;进样量为10ul;分析时间为80min。进而对主要色谱峰的相对保留时间和相对峰面积等指纹图谱参数进行分析,结果表明各相关指标符合图谱的评价标准,为沙枣果实的指纹图谱研究深入开展沙枣种质资源合理开发利用提供理论依据。该法操作简单,重复性好,可行性高。