PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori
PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori
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DOI：
10.1073/pnas.95.22.13108
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发表时间：
1998-10-27
影响因子：
11.1
通讯作者：
Berg, DE
中科院分区：
文献类型：
--
作者：
Akopyants, NS;Fradkov, A;Berg, DE
仅具有细菌物种的某些菌株特征的基因可能具有很大的生物学意义。在这里，我们描述了一种基于 PCR 的消减杂交方法，可有效检测此类 DNA 并将其应用于胃病原体幽门螺杆菌，通过与基因组已完全测序的不相关菌株 (26695) 进行消减杂交，获得了 18 个猴定植菌株 (J166) 特异的 DNA，7 个 J166 特异性克隆与 26695 基因组没有 DNA 序列匹配，其他 11 个克隆混合在一起，相邻的补丁与 26695 中的任何序列匹配或不匹配。在蛋白质水平上，se-yen 克隆与假定的 DNA 限制性修饰酶具有同源性，并且两个克隆与假定的代谢酶具有同源性。另外九个与指定功能的蛋白质没有数据库匹配。通过使用 12 个消减克隆特异的引物和互补 Southern 印迹杂交对 13 个不相关的幽门螺杆菌菌株进行 PCR 测试，表明这些 DNA 在幽门螺杆菌群体中具有高度多态性，每个菌株产生不同的基因特异性 PCR 扩增模式。如本文所述，对多态性 DNA 的搜索应有助于识别病原体中以前未知的毒力基因，并为微生物遗传多样性和进化提供新的见解。
Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori, Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695), Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695, At the protein level, se-yen clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H, pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.