Zietkiewicz E, Rafalski A, Labuda D
Hôpital Ste-Justine, Département de Pédiatrie, Université de Montréal, Quebec, Canada.
Genomics. 1994 Mar 15;20(2):176-83. doi: 10.1006/geno.1994.1151.
Simple sequence repeats (SSR), or microsatellites, are ubiquitous in eukaryotic genomes. Here we demonstrate the utility of microsatellite-directed DNA fingerprinting by polymerase chain reaction (PCR) amplification of the interrepeat region. No sequencing is required to design the oligonucleotide primers. We tested primers anchored at 3' or 5' termini of the (CA)n repeats, extended into the flanking sequence by 2 to 4 nucleotide residues [3'-anchored primers: (CA)8RG, (CA)8RY, and (CA)7RTCY; and 5'-anchored primers: BDB(CA)7C, DBDA(CA)7, VHVG(TG)7 and HVH(TG)7T]. Radioactively labeled amplification products were analyzed by electrophoresis, revealing information on multiple genomic loci in a single gel lane. Complex, species-specific patterns were obtained from a variety of eukaryotic taxa. Intraspecies polymorphisms were also observed and shown to segregate as Mendelian markers. Inter-SSR PCR provides a novel fingerprinting approach applicable for taxonomic and phylogenetic comparisons and as a mapping tool in a wide range of organisms. This application of (CA)n repeats may be extended to different microsatellites and other common dispersed elements.
简单序列重复（SSR），即微卫星，在真核生物基因组中普遍存在。在此，我们通过对重复序列间区域进行聚合酶链反应（PCR）扩增，展示了微卫星定向DNA指纹图谱的实用性。设计寡核苷酸引物无需进行测序。我们测试了锚定在（CA）n重复序列3'或5'末端的引物，这些引物延伸到侧翼序列2至4个核苷酸残基处[3'锚定引物：（CA）8RG、（CA）8RY和（CA）7RTCY；5'锚定引物：BDB（CA）7C、DBDA（CA）7、VHVG（TG）7和HVH（TG）7T]。通过电泳分析放射性标记的扩增产物，可在单一凝胶泳道中揭示多个基因组位点的信息。从多种真核生物分类群中获得了复杂的、物种特异性的模式。还观察到种内多态性，并显示其作为孟德尔标记进行分离。SSR间PCR提供了一种新的指纹图谱方法，适用于分类学和系统发育比较，并可作为多种生物的图谱绘制工具。（CA）n重复序列的这种应用可能扩展到不同的微卫星和其他常见的分散元件。
