A rapid PCR-based method for genetically mapping ESTs
A rapid PCR-based method for genetically mapping ESTs
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DOI：
10.1007/s001220051646
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发表时间：
2001-02-01
影响因子：
5.4
通讯作者：
Richardson, TE
中科院分区：
文献类型：
--
作者：
Cato, SA;Gardner, RC;Richardson, TE
开发了一种简单的半自动化程序，用于将表达序列标签 (EST) 转换为可定位的遗传标记。聚合酶链式反应用于扩增紧邻基因编码区 5' 或 3' 的区域，以最大限度地提高等位基因之间的序列变异性。扩增等位基因中的片段长度和核苷酸取代多态性可以使用溴化乙锭染色或自动激光荧光来检测。使用 ABI 377 DNA 测序仪分析 6% 非变性丙烯酰胺凝胶，证明能够解析包含核苷酸取代的扩增等位基因之间形成的同源双链体和异源双链体，以及解析等位基因长度差异。通过这种方法，来自一系列松属物种的 60 个 EST 中的 75% 可以在辐射松和火炬松的三个谱系中的每一个中进行基因图谱绘制。此外，在每个家系中检测到 42% 的 EST 标记有 3 个或 4 个等位基因。
A simple, semi-automatable procedure was developed for converting expressed sequence tags (ESTs) into mappable genetic markers, The polymerase chain reaction is used to amplify regions immediately 5' or 3' to the coding regions of genes in order to maximise sequence variability between alleles. Fragment length and nucleotide substitution polymorphisms among amplified alleles can be detected using either ethidium bromide staining or automated laser-based fluorescence. A 6% non-denaturing acrylamide gel, analysed with an ABI 377 DNA sequencer, proved capable of resolving homoduplexes and heteroduplexes formed between amplified alleles containing nucleotide substitutions as well as resolving allelic length differences. With this approach 75% of 60 ESTs from a range of Pinus species could be genetically mapped in each of three pedigrees from P. radiata and P. taeda. Furthermore, three or four alleles were detected in each pedigree for 42% of the EST markers.