Hauser M T, Adhami F, Dorner M, Fuchs E, Glössl J
Center of Applied Genetics, University of Agricultural Sciences Vienna, Austria.
Plant J. 1998 Oct;16(1):117-25. doi: 10.1046/j.1365-313x.1998.00271.x.
Rapid and efficient procedures for the detection of sequence polymorphisms are essential for chromosomal walking and mutation detection analyses. While DNA chip technology and denaturing high-performance liquid chromatography (DHPLC) are the methods of choice for large scale facilities, small laboratories are dependent on simple ready-to-use techniques. We show that heteroduplex analysis on high resolution gel matrices efficiently detects sequence polymorphism differing as little as a single base pair (e.g. single-nucleotide polymorphism, SNP) with standard laboratory equipment. Furthermore, the matrices also discerned differences between homoduplexes, a prerequisite for co-dominant markers. The markers thus generated are referred to as duplex analysis markers. We designed PCR primers for 36 Arabidopsis thaliana loci ranging in length from 230 bp to 1000 bp. Among three ecotypes, more than half (n = 19) of the loci examined were polymorphic; five of which contained three different alleles. This simple, high resolution technique can be used to rapidly convert sequence tagged sites into co-dominant PCR-based molecular markers for fine-scale mapping studies and chromosomal walking strategies as well as for the detection of mutations in particular genes.
用于检测序列多态性的快速高效方法对于染色体步移和突变检测分析至关重要。虽然DNA芯片技术和变性高效液相色谱法（DHPLC）是大型机构的首选方法，但小型实验室依赖于简单易用的技术。我们表明，使用标准实验室设备，在高分辨率凝胶基质上进行异源双链分析能够有效检测出差异小至单个碱基对的序列多态性（例如单核苷酸多态性，SNP）。此外，这些基质还能区分同源双链之间的差异，这是共显性标记的一个先决条件。由此产生的标记被称为双链分析标记。我们为36个拟南芥基因座设计了长度在230 bp至1000 bp之间的PCR引物。在三种生态型中，超过一半（n = 19）的检测基因座是多态性的；其中五个包含三种不同的等位基因。这种简单的高分辨率技术可用于快速将序列标签位点转化为基于共显性PCR的分子标记，用于精细定位研究和染色体步移策略，以及检测特定基因中的突变。
