Plant and Soil Science Department, Montana State University, 59717, Bozeman, MT, USA.
Theor Appl Genet. 1992 Sep;84(7-8):1002-8. doi: 10.1007/BF00227417.
Speed, efficiency, and safety considerations have led many genome mapping projects to evaluate polymerase chain reaction (PCR) sequence amplification as an alternative to Southern blot analysis. However, the availability of informative primer sequences can be a limiting factor in PCR-based mapping. An alternative to random amplified polymorphism detection (RAPD) is the sequence-tagged-site (STS) approach. If informative primer sequences could be derived from known sequences, then current maps, which are based on both known function and anonymous clones, might be easily converted to maps utilizing PCR technology. In this paper, four pairs of primer sequences were obtained from published sequences, and four pairs were obtained by sequencing portions of DNA clones from genomic clones derived from a random genomic library used in the North American Barley Genome Mapping Project (NABGMP). These primers were used to screen for polymorphisms in the progeny of a winter x spring and a spring x spring barley cross. Two types of polymorphisms were distinguished using these primer sets: (1) insertion/deletion events that could be read directly from agarose gels, and (2) point mutation events. The latter were identified using polyacrylamide-gel electrophoresis of PCR products following digestion with restriction endonucleases (four-base cutters). To determine whether the PCR-based polymorphisms were allelic to polymorphisms identified by the clones from which the primer sequences derived, chromosomal assignments and (when possible) co-segregation analysis was performed.
速度、效率和安全考虑因素促使许多基因组图谱绘制项目评估聚合酶链反应（PCR）序列扩增作为 Southern 印迹分析的替代方法。然而，信息引物序列的可用性可能是基于 PCR 的图谱绘制的一个限制因素。随机扩增多态性检测（RAPD）的替代方法是序列标记位点（STS）方法。如果可以从已知序列中获得信息引物序列，那么当前基于已知功能和匿名克隆的图谱可能很容易转换为利用 PCR 技术的图谱。在本文中，从已发表的序列中获得了四对引物序列，从北美大麦基因组图谱项目（NABGMP）中使用的随机基因组文库衍生的基因组克隆的 DNA 克隆的部分测序中获得了四对引物序列。这些引物用于筛选冬小麦和春小麦杂交后代的多态性。使用这些引物集区分了两种类型的多态性：（1）可以直接从琼脂糖凝胶读取的插入/缺失事件，和（2）点突变事件。后者通过用限制内切酶（四碱基切割酶）消化 PCR 产物的聚丙烯酰胺凝胶电泳来鉴定。为了确定基于 PCR 的多态性是否与引物序列衍生的克隆所鉴定的多态性等位，进行了染色体分配和（如果可能）共分离分析。
