Improved DNA fragment length estimation in capillary electrophoresis
Improved DNA fragment length estimation in capillary electrophoresis
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DOI：
10.1002/elps.200700523
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发表时间：
2008-03-01
期刊：
影响因子：
2.9
通讯作者：
Jakobsen, Kjetill S.
中科院分区：
文献类型：
--
作者：
Akbari, Akbar;Marthinsen, Gunnhild;Jakobsen, Kjetill S.
在毛细管电泳法中，带电的DNA链在穿过毛细管时根据片段长度进行分级，因为较短的DNA片段通过筛分基质的速度更快。多重内部大小标准被用来估计未知DNA片段的大小。在文献中，有关于GeneScan-500(GS500)内部大小标准中250和340个核苷酸片段迁移异常的陈述。这种大小标准的异常迁移显然会在未知碎片的估计中引入误差。因此，许多分析程序简单地排除了其中的一些片段。在目前的工作中，我们首先评估在CE中使用的内部尺寸标准中排除每个碎片的效果。其次，提出了一种基于异常碎片真值估计的方法。通过新方法获得的结果表明，与在估计未知DNA片段的大小时排除或包括GS500中的一个(或两个)异常片段所获得的结果相比，有显著的改善。在高分子量区，平均误差从ABI GeneMapper的1.91个碱基(不包括250个碱基)减少到新方法的0.15个碱基(使用250和340个碱基的估计值)。在低分子量区，排除这两个片段将使结果比ABI GeneMapper平均提高0.74个碱基对。
In CE the charged DNA strands are fractionated according to fragment lengths as they migrate through the capillary, since shorter DNA fragments pass through the sieving matrix faster. Multiplexed internal size standards are used to estimate the size of unknown DNA fragments. In the literature there are statements about migration abnormalities for the 250 and 340 bp fragments in the GeneScan-500 (GS500) internal size standards. Such anomalous migration of size standards could obviously introduce errors in the estimation of unknown fragments. Therefore, a number of analysis programs simply exclude some of these fragments. In the present work we first evaluate the effect of excluding each of the fragments in the internal size standards used in CE. Next, a method which is based on estimating the true values of the anomalous fragments is presented. The results obtained by the new method indicate a significant improvement compared to results obtained when one (or both) of the anomalous fragments in GS500 is (are) excluded or included when estimating the size of unknown DNA fragments. In the higher-molecular-weight region, the average error is reduced from 1.91 bp in ABI GeneMapper (excluding 250 bp) to 0.15 bp in the new method (using the estimated values for 250 and 340 bp). In the lower-molecular-weight region, excluding both fragments will improve the results by an average of 0.74 bp compared to ABI GeneMapper.