Lin Ming-Tseh, Rich Roy G, Shipley Royce F, Hafez Michael J, Tseng Li-Hui, Murphy Kathleen M, Gocke Christopher D, Eshleman James R
The Sol Goldman Pancreatic Cancer Research Center, Departments of Pathology and Oncology, Johns Hopkins School of Medicine, 1550 Orleans Street, Baltimore, MD 21231, USA.
J Mol Diagn. 2007 Nov;9(5):598-603. doi: 10.2353/jmoldx.2007.070022. Epub 2007 Oct 4.
Several methods exist to retrieve and purify DNA fragments after agarose or polyacrylamide gel electrophoresis for subsequent analyses. However, molecules present in low concentration and molecules similar in size to their neighbors are difficult to purify. Capillary electrophoresis has become popular in molecular diagnostic laboratories because of its automation, excellent resolution, and high sensitivity. In the current study, the ABI Prism 310 Genetic Analyzer was reconfigured into a fraction collector by adapting the standard gel block to accommodate a collection tube at the distal end of capillary. The time to collect the desired peaks was estimated by extrapolating from standard capillary electrophoresis using the original gel block. Fraction collection from a mixture of DNA fragments amplified from wild type and several internal tandem duplication mutations of the FMS-like tyrosine kinase 3 (Flt3) gene yielded highly purified DNA fragments containing internal tandem duplication mutations and predictable electrokinetics using the reconstructed gel block. The reconfigured instrument could successfully isolate DNA amplicons from extremely low-amplitude peaks (110 relative fluorescent units), which were undetectable using polyacrylamide gel electrophoresis. In addition, we successfully isolated bands that were only three bases apart that comigrated on polyacrylamide gel electrophoresis. DNA sequencing was used to confirm that the correct peaks were recovered at sufficient purity.
在琼脂糖或聚丙烯酰胺凝胶电泳后，有几种方法可用于回收和纯化DNA片段以便后续分析。然而，低浓度存在的分子以及大小与其相邻分子相似的分子很难纯化。毛细管电泳因其自动化、出色的分辨率和高灵敏度，在分子诊断实验室中已变得很流行。在当前研究中，通过调整标准凝胶块以容纳毛细管末端的收集管，将ABI Prism 310基因分析仪重新配置为一个馏分收集器。通过使用原始凝胶块从标准毛细管电泳进行外推，估算收集所需峰的时间。从野生型以及FMS样酪氨酸激酶3（Flt3）基因的几个内部串联重复突变体扩增得到的DNA片段混合物中进行馏分收集，使用重构的凝胶块可得到高度纯化的含有内部串联重复突变的DNA片段以及可预测的动电现象。重新配置的仪器能够成功地从极低振幅峰（110相对荧光单位）中分离出DNA扩增子，而使用聚丙烯酰胺凝胶电泳则无法检测到这些峰。此外，我们成功地分离出了在聚丙烯酰胺凝胶电泳中迁移在一起、仅相差三个碱基的条带。使用DNA测序来确认回收的是纯度足够的正确峰。
