Kline M C, Redman J W, Reeder D J, Duewer D L
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Appl Theor Electrophor. 1996;6(1):33-41.
The 100-1,000 basepair size typical of PCR-amplified DNA fragments demands high resolution electrophoretic gels for the adequate characterization of small differences among samples. We have studied the behavior of a number of commercial sizing ladders in three classes of separation systems: polyacrylamides with discontinuous buffer, proprietary acrylamides with continuous buffer, and agarose-like materials with continuous buffer. None of the ladders examined perform adequately in any of these systems using vendor-supplied nominal ladder component basepair sizes. All ladders successfully typed D1S8O alleles after calibration with the allelic ladder (replacing the nominal size values with the least squares estimate of allele/matrix-specific apparent sizes). Some ladders and matrices are qualitatively better than others. No one ladder proved consistently better than others; a polyacrylamide gel with ribose modifier provided the most precise results in this study. Appropriately calibrated electrophoretic apparent sizes must be used for results to be validly exchanged among laboratories. Appropriate allelic ladders or a well defined subset of known alleles can serve as the calibration system.
聚合酶链反应（PCR）扩增的DNA片段通常大小在100至1000个碱基对之间，这就需要高分辨率的电泳凝胶，以便充分表征样品之间的微小差异。我们研究了多种商业尺寸标准物在三类分离系统中的表现：具有不连续缓冲液的聚丙烯酰胺、具有连续缓冲液的专利丙烯酰胺以及具有连续缓冲液的琼脂糖类材料。在所研究的这些系统中，使用供应商提供的标准尺寸标准物碱基对大小，没有一种标准物能表现良好。在用等位基因标准物校准后（用等位基因/基质特异性表观大小的最小二乘估计值取代标称大小值），所有标准物都成功地对D1S8O等位基因进行了分型。有些标准物和基质在质量上比其他的更好。没有一种标准物被证明始终比其他的更好；在本研究中，带有核糖修饰剂的聚丙烯酰胺凝胶提供了最精确的结果。为了使结果能在各实验室之间有效交流，必须使用经过适当校准的电泳表观大小。合适的等位基因标准物或一组明确界定的已知等位基因子集可作为校准系统。
