Application of an improved DNA gene extraction method based on magnetic beads in conventional HLA sequence-based typing
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摘要: 目的: 建立并改良磁珠提取基因DNA的方法,以应用于HLA常规测序分型。方法: 使用磁珠法自动提取系统从全血中提取基因组DNA,利用DNA定量仪测定DNA浓度与纯度,利用测序仪对PCR扩增产物进行测序及相关的分析软件进行HLA分型结果分析。比较改良前后DNA浓度与纯度、HLA分型结果成功率、信噪比及测序胶图的反应格局。结果: 改良后96份DNA浓度的均一性、纯度均优于改良前;32份HLA分型结果中HLA-A、B位点成功率改良后高于改良前,差异有统计学意义(P<0.01);改良后32人份HLA-B第二外显子信噪比(N/S)合格率均高于改良前,差异有统计学意义(P<0.01);改良后32份分型胶图反应格局均优于改良前。结论: 改良磁珠提取DNA法能稳定、可靠地应用于HLA测序分型,具有快速、高通量化的特点和广泛的应用前景。Abstract: Objective: To establish and improve the DNA gene extraction method based on magnetic beads for HLA sequence-based typing. Method: Genomic DNA was extracted from the whole blood samples using magnetic beads method through automatic extraction system. The concentration and purity of DNA were determined through the DNA quantitative instrument; the PCR amplified products were sequenced through the sequencer. HLA typing results were analyzed by the related analysis software. Result: The homogeneity of concentration and purity of DNA extracted by the improved method in 96 copies, were better than those extracted by the traditional DNA gene extraction method. Both the success rate of the HLA genotyping on HLA-A, B locus and the experiment's qualified rate based on HLA-B Exon2 signal-to-noise ratio(N/S) in 32 copies were higher than those using normal DNA gene extraction method statistically(P<0.01). The reaction patterns of the PCR products' electrophoretogram were also improved than the novel method. Conclusion: This improved DNA gene extraction method based on magnetic beads is proved to be a more stable,reliable and high throughtput DNA gene extraction method for HLA sequence-based typing, which will be meaningful for DNA genotyping.
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Key words:
- Deoxyribonucleic acid /
- specimen /
- genotyping /
- sequence-based typing
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