Application of automated DNA sizing technology for genotyping microsatellite loci.
Application of automated DNA sizing technology for genotyping microsatellite loci.
复制标题
应用自动 DNA 大小测定技术对微卫星位点进行基因分型。
DOI：
10.1016/s0888-7543(05)80126-0
复制
发表时间：
1992
期刊：
影响因子：
4.4
通讯作者：
Diehl,SR
中科院分区：
文献类型：
--
作者：
Ziegle,JS;Su,Y;Corcoran,KP;Nie,L;Mayrand,PE;Hoff,LB;McBride,LJ;Kronick,MN;Diehl,SR
高度多态的微卫星位点为基因定位研究提供了巨大的希望，但实现这一潜力将需要在准确和有效的基因分型方法上进行实质性的改进。在这里，我们报告了一种基于荧光标记PCR引物的基因分型方法，并使用自动DNA片段分析仪对PCR产物进行大小表征。我们利用三种不同的荧光染料的可用性来标记大小重叠的独特基因座，这一创新使可以同时分析的基因座数量增加了三倍。我们用第四种染料标记尺寸标准，并将其与微卫星PCR产物结合在每个凝胶通道中。计算机程序提供了非常快速和准确的微卫星等位基因大小和有效的数据管理。此外，荧光信号在更大的强度范围内是线性的，而不是传统的放射自显影。这有利于基因座的多路复用（因为信号强度通常变化很大），并有助于区分主峰和伪峰，从而提高基因分型的准确性。
Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.