Bailly Xavier, Béna Gilles, Lenief Vanina, de Lajudie Philippe, Avarre Jean-Christophe
Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR113 IRD/INRA/CIRAD/UM2/Agro-M, Campus International de Baillarguet, Montpellier F34398, France.
J Microbiol Methods. 2006 Oct;67(1):114-24. doi: 10.1016/j.mimet.2006.03.006. Epub 2006 Apr 19.
Some bacterial species, like nitrogen-fixing Sinorhizobium that interact with Medicago plants, are prone to frequent horizontal gene transfers. Investigation of their genetic structure requires to study polymorphism patterns at many loci. Although DNA microarrays represent a method of choice for high throughput analysis of polymorphisms, this technology yet remains an expensive and heavy approach, thus depriving most of research groups from this powerful tool. In an attempt to overcome this limitation, we have developed a simple genotyping procedure by DNA microarrays, and have evaluated its ability to characterize a Sinorhizobium population. Thirty 18- to 24-mer oligonucleotide probes were designed to target the most frequent mutations in three polymorphic loci of Sinorhizobium meliloti and S. medicae. Probe hybridization efficiency was compared on two spotting surfaces: nylon membranes and epoxy-coated glass slides. Epoxy-coated glass slides revealed more sensitive than nylon membranes and allowed discrimination of single mismatches. Using this procedure, an uncharacterized population consisting of 33 S. meliloti/S. medicae isolates was successfully genotyped.
一些细菌物种，比如与苜蓿属植物相互作用的固氮中华根瘤菌，容易频繁发生水平基因转移。对它们的遗传结构进行研究需要在多个位点研究多态性模式。尽管DNA微阵列是多态性高通量分析的首选方法，但这项技术仍然是一种昂贵且繁琐的方法，因此大多数研究团队无法使用这种强大的工具。为了克服这一限制，我们开发了一种通过DNA微阵列进行简单基因分型的程序，并评估了其对根瘤菌群体进行特征描述的能力。设计了30个18至24个碱基的寡核苷酸探针，以靶向苜蓿中华根瘤菌和中慢生根瘤菌三个多态性位点中最常见的突变。在两种点样表面上比较了探针杂交效率：尼龙膜和环氧涂层载玻片。环氧涂层载玻片比尼龙膜更灵敏，能够区分单个错配。使用这个程序，成功地对由33株苜蓿中华根瘤菌/中慢生根瘤菌分离株组成的未鉴定群体进行了基因分型。
