Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing.
Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing.
复制标题
合成用于自动片段大小测定的荧光内部 DNA 标准品的经济有效的方法。
DOI：
10.2144/01314st06
复制
发表时间：
2001
期刊：
影响因子：
2.7
通讯作者：
D. Grattapaglia
中科院分区：
文献类型：
--
作者：
R. Brondani;D. Grattapaglia
我们描述了一种简单且经济有效的方法，用于使用自动 DNA 测序仪合成用于片段大小的内部荧光标记 DNA 标准品。开发了一组用 ROX 标记的引物对来扩增源自植物叶绿体 DNA 保守区域的 12 个 DNA 片段（58-417 bp）。这些扩增片段混合在一起，构成荧光内部 DNA 大小标记。通过使用合成的尺寸标准品和商业内部尺寸标准品 GeneScan Rox500 估计在四个二核苷酸微卫星位点扩增的 20 个等位基因的大小来评估尺寸标准品的精度。进行了多次凝胶内和凝胶间比较，并进行了方差分析。使用合成 DNA 标准品和商业标准品获得的大小估计值之间没有观察到显着差异。这种基于 PCR 的简便通用方法用于合成内标，可以在使用微卫星或 AFLP 标记实施大型基因分型实验时显着节省成本。
We describe a simple and cost-effective method for the synthesis of an internal fluorescently labeled DNA standard for fragment sizing using an automated DNA sequencer. A set of primer pairs labeled with ROX was developed to amplify 12 DNA fragments, 58-417 bp, derived from a conserved region of plant chloroplast DNA. These amplified fragments were mixed together, constituting a fluorescent internal DNA size marker. The precision of the size standard was evaluated by estimating the size of 20 alleles that were amplified at four dinucleotide microsatellite loci with the synthesized size standard and the commercial internal sizing standard, GeneScan Rox500. A number of intra-gel and inter-gel comparisons were run, and an analysis of variance was carried out. No significant difference was observed between the size estimates obtained with the synthesized DNA standard and the commercial standard. This facile and general PCR-based method for the synthesis of internal standards allows for significant savings in the implementation of large genotyping experiments using microsatellite or AFLP markers.