Comparison of fluorescence-based semi-automated genotyping of multiple microsatellite loci with autoradiographic techniques.
Comparison of fluorescence-based semi-automated genotyping of multiple microsatellite loci with autoradiographic techniques.
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多个微卫星位点基于荧光的半自动基因分型与放射自显影技术的比较。
DOI：
10.1006/geno.1994.1344
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发表时间：
1994
期刊：
影响因子：
4.4
通讯作者：
Levitt,RC
中科院分区：
文献类型：
--
作者：
Schwengel,DA;Jedlicka,AE;Nanthakumar,EJ;Weber,JL;Levitt,RC
基于聚合酶链反应的微卫星标记半自动基因分型的高效荧光方法的实际应用将取决于提供准确和可重复数据的可靠方案的发展。在本报告中，我们比较了基于荧光的方案与基准放射标记方法的准确性，该方法依赖于已知的序列阶梯或来自参考个体的扩增DNA，通过放射自显影进行分级。三个微卫星标记，IGF1 (mfd 1), D4S174 （mfd 59）和D5S211 (mfd 154)，产品尺寸重叠，每个标记用不同的荧光团标记，并在单个电泳通道中与内部尺寸标准同时运行。通过使用这两种技术对5个较大的CEPH家族（884、1331、1332、1333和1362）的每个等位基因的大小进行比较。在462个可能的等位基因中，两种方法比较发现了4个差异（0.8%）。我们得出的结论是，基于荧光的方案至少与标准放射性标记技术一样准确，因为没有任何尺寸误差是由于基于荧光的技术而产生的。我们描述了这种基于荧光的方案的适应性，以同时分析每个电泳通道多达24个微卫星位点。这些高度精确和高效的半自动化技术将在高分辨率基因组分析中发挥作用。
The practical application of highly efficient fluorescence-based methods for the semi-automated genotyping of polymerase chain reaction-based microsatellite markers will depend on the development of robust protocols that provide accurate and reproducible data. In the present report we compare the accuracy of a fluorescence-based protocol with a benchmark radiolabeling method that depends on a known sequence ladder or amplified DNA from reference individuals for sizing by autoradiography. Three microsatellite markers, IGF1 (mfd 1), D4S174 (mfd 59), and D5S211 (mfd 154), with products overlapping in size were each labeled with a different fluorophore and run simultaneously with an internal size standard in a single electrophoretic lane. The size of each allele was compared for these markers by using both techniques for five larger CEPH families (884, 1331, 1332, 1333, and 1362). Of 462 possible alleles, four discrepancies (0.8%) were identified when the two approaches were compared. We conclude that the fluorescence-based protocol is at least as accurate as the standard radiolabeling technique since none of the sizing errors arose as a result of the fluorescence-based technique. We describe the adaptation of this fluorescence-based protocol to the simultaneous analysis of up to 24 microsatellite loci per electrophoretic lane. These highly accurate and efficient semi-automated techniques will be useful in high-resolution genomic analyses.