McEvoy C R, Seshadri R, Firgaira F A
Flinders University of South Australia.
Biotechniques. 1998 Sep;25(3):464-70.
We have investigated the potential of the PE Applied Biosystems Model 373 Automated DNA Sequencer and GENESCAN software to size minisatellite alleles ranging in size from 230 bp to 2.5 kbp. We report on the use of a native (non-denaturing) acrylamide gel system and fluorescent dUTP labeling of PCR products. The observed variability in size calling ranged from +/- 0.4-bp standard deviation (SD) at the lower end of the size range to +/- 37.5-bp SD for the largest allele. Both within-gel and between-gel variability in sizing increased with larger alleles, in particular when sizes exceeded 2 kbp. Size-calling differences were observed dependent on the method used to fluorescently label the PCR products and with the fluorescent dye type and concentration used in incorporation. The benefits and limitations of the current GENESCAN software in sizing large DNA fragments are also discussed.
我们研究了应用生物系统公司373型自动DNA测序仪和GENESCAN软件对大小在230 bp至2.5 kbp之间的微卫星等位基因进行大小测定的潜力。我们报告了使用天然（非变性）聚丙烯酰胺凝胶系统和对PCR产物进行荧光dUTP标记的情况。观察到的大小测定变异性范围为：在大小范围下限处标准偏差（SD）为±0.4 bp，对于最大的等位基因，标准偏差为±37.5 bp。随着等位基因增大，凝胶内和凝胶间大小测定的变异性均增加，尤其是当大小超过2 kbp时。观察到大小测定差异取决于用于对PCR产物进行荧光标记的方法以及掺入时使用的荧光染料类型和浓度。还讨论了当前GENESCAN软件在测定大DNA片段大小时的优点和局限性。
