Jabasini Mohammad, Zhang Lihua, Dang Fuquan, Xu Feng, Almofli Mohamad R, Ewis Ashraf A, Lee Juwon, Nakahori Yutaka, Baba Yoshinobu
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima, CREST, Japan Science and Technology Corporation, Shomachi, Tokushima, Japan.
Electrophoresis. 2002 May;23(10):1537-42. doi: 10.1002/1522-2683(200205)23:10<1537::AID-ELPS1537>3.0.CO;2-E.
Validation of microchip electrophoresis in DNA analysis has been carried out using an Agilent 2100 Bioanalyzer. With a DNA 500 Assay Kit, the reproducibility and accuracy of fragment sizing of a 10 bp DNA ladder have been shown to be satisfactory with the relative standard deviation and the relative error mostly below 1.0 and 5.0% (n = 12), respectively. Both intraday and interday validations of fragment sizing and quantitation have also been performed with a 7500 Assay Kit (n = 48). Although the results of quantitation are not as good as that of sizing, due to the manual introduction of samples and markers into the chip wells, they are still sufficient to carry out further analyses of practical samples. Based on such reliable results, fast analysis of DNA polymorphisms on the human Y-chromosome has been realized with microchip electrophoresis. The total analysis times of three genomic polymorphisms on the Y-chromosome, Y Alu polymorphism, 47z/StuI, and 12f2, are all within 100 s, and the relative standard deviation and relative error of fragment sizes are below 3.5 and 3.7%, respectively. In addition, a mixture of nine DNA markers on the human Y-chromosome related to examine the cause of spermatogenic failure have been separated successfully with the smallest fragment size difference of 7 bp. Our results demonstrate the potential of microchip electrophoresis in polymorphism analysis with the advantages of high speed, good reproducibility, high precision, and high resolution.
已使用安捷伦2100生物分析仪对微芯片电泳在DNA分析中的性能进行了验证。使用DNA 500分析试剂盒，10 bp DNA梯状条带片段大小测定的重现性和准确性令人满意，相对标准偏差和相对误差大多分别低于1.0%和5.0%（n = 12）。还使用7500分析试剂盒进行了片段大小测定和定量的日内和日间验证（n = 48）。尽管定量结果不如大小测定结果好，这是由于手动将样品和标记物引入芯片孔中，但这些结果仍足以对实际样品进行进一步分析。基于这些可靠的结果，利用微芯片电泳实现了对人类Y染色体上DNA多态性的快速分析。Y染色体上三种基因组多态性，即Y Alu多态性、47z/StuI和12f2的总分析时间均在100秒以内，片段大小的相对标准偏差和相对误差分别低于3.5%和3.7%。此外，成功分离了与检查生精失败原因相关的人类Y染色体上的九种DNA标记物混合物，最小片段大小差异为7 bp。我们的结果证明了微芯片电泳在多态性分析中的潜力，具有高速、良好的重现性、高精度和高分辨率的优点。
