Ellison J S
National Institutes of Health, National Center for Human Genome Research, Bethesda, MD 20892.
Mol Biotechnol. 1996 Feb;5(1):17-31. doi: 10.1007/BF02762409.
Conventional SSCP analysis of DNA amplified by polymerase chain reaction (PCR-SSCP) is one of the simplest and most reliable tools for identifying point mutations, and small insertions or deletions. The sensitivity of the technique is increased by using the Applied Biosystems (ABI) semiautomated DNA sequencer equipped with GENESCAN 672 software for F-SSCP. The four-dye ABI system permits a red dye-labeled internal lane standard to be run in the same lanes as the DNA being examined, leaving three dye colors for labeling DNA of interest. The internal lane standard is used to normalize gels or correct for minor differences in apparent electrophoretic mobility between lanes. Correction for these lane-dependent differences in migration and the capability to stack data from two different lanes on the computer screen makes it possible to detect sequence variants that produce very small mobility shifts. Coelectrophoresis of control and unknown DNA in the same lane, using different dye labels for each, is also helpful for detecting sequence variants that produce small mobility changes. Multiplexing multiple F-SSCP targets in the same lane increases sample throughput.
聚合酶链反应扩增DNA的传统单链构象多态性分析（PCR - SSCP）是识别点突变以及小的插入或缺失的最简单且最可靠的工具之一。通过使用配备用于F - SSCP的GENESCAN 672软件的应用生物系统公司（ABI）半自动DNA测序仪，可提高该技术的灵敏度。四染料ABI系统允许将红色染料标记的内标泳道与被检测的DNA在同一泳道中运行，这样就剩下三种染料颜色用于标记感兴趣的DNA。内标泳道用于使凝胶标准化或校正泳道之间表观电泳迁移率的微小差异。校正这些与泳道相关的迁移差异以及在计算机屏幕上叠加来自两个不同泳道数据的能力，使得检测产生非常小的迁移率变化的序列变异成为可能。在同一泳道中对对照DNA和未知DNA进行共电泳，对每种DNA使用不同的染料标记，也有助于检测产生小迁移率变化的序列变异。在同一泳道中对多个F - SSCP靶标进行多重分析可提高样品通量。
