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优化了S1核酸酶突变检测体系,结果表明(1)扩增法比杂交法产生异源双链DNA更节省时间;(2)PCR产物可以直接作为突变检测的底物;(3)适当降低反应温度、适当增加反应缓冲液中的NaCl浓度、适当缩短反应时间可提高突变检测效果。 相似文献
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为解析二化螟Chilo suppressalis体内参与降解双链RNA(double-stranded RNA,dsRNA)的关键核酸酶的功能,克隆二化螟不同的非专一性核酸酶(non-specific nuclease,NUC)基因,并对这些基因进行生物信息学分析和组织定量表达分析,同时对dsRNA降解酶(dsRNA degrading nuclease,dsRNase)活力的组织分布进行研究。结果显示,共克隆获得5个NUC基因,其中有4个编码dsRNase亚家族基因(CsdsRNase1~CsdsRNase4)和1个编码Endonuclease G亚家族基因(CsEndoG)。5个NUC基因的开放阅读框核苷酸序列长度范围为828~1 338 bp,编码275~445个氨基酸残基,其分子量大小为31.68~49.57 kD,预测等电点为5.48~9.42。CsdsRNase1和CsdsRNase2含有信号肽序列,两者相似度极高,且与家蚕Bombyx mori和斜纹夜蛾Spodoptera litura中具有dsRNA降解酶活力的dsRNase同源聚类;CsdsRNase1和CsdsRN... 相似文献
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Enzymoserological comparison of a selection of leptospira strains tested with sera from rabbits immunized with unpurified DNase of Leptospira interrogans, serotype canicola, indicates the production of DNase of serologically very similar properties by the serotypes canicola, autumnalis, icterohemorrhagiae and pomona. The DNase produced by serotype hyos was serologically different from the others, while the serotypes grippotyphosa and bataviae did not produce DNases at all. The method used made it possible to differentiate between leptospiral DNase and normally occurring DNases in the serum samples. Neither leptospira DNase nor specific leptospira-DNase-antibodies could be detected in dog sera with high agglutinationlysis titres after natural infection. 相似文献
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[目的]构建细菌性条斑病菌诱导下的水稻抗病均一化差减c DNA文库,对获得的差异表达ESTs进行生物信息学及抗病相关基因的表达分析。[方法]以水稻IR26(Tester)和两优培九(Driver)幼苗5~6叶期叶片为材料,采用双链特异性核酸酶(DSN)介导的均一化消减杂交技术,构建细菌性条斑病菌JH01诱导的水稻抗病相关基因差减c DNA文库,以p LB-F和p LB-R为引物,通过菌液PCR扩增对差减文库的质量进行检测。[结果]c DNA文库的插入片段分布在0.5~2.0 kb,平均大小约为1.0 kb,重组率达95%。序列测定分析发现,随机挑取的504个克隆中有98条差异表达基因;经BLAST比对和GO注释发现,59条序列具有同源基因,分别参与信号转导、能量代谢、过敏性坏死反应、防卫反应等;另外39条序列无相似基因,有待进一步研究。通过实时荧光定量PCR发现,从该文库中分离得到的Os NDPK4参与细菌性条斑病菌JH01诱导的水稻防卫反应。[结论]水稻受细菌性条斑病菌侵染的c DNA文库质量较好,为研究条斑病菌致病性因子与水稻互作机制奠定基础。 相似文献
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Michel A. Salmon Marina Vendrame Jean Kummert Philippe Lepoivre 《European journal of plant pathology / European Foundation for Plant Pathology》2002,108(8):755-762
Real-time PCR with fluorogenic hydrolysis probes (5' nuclease assay) is increasingly used for the detection of pathogens for diagnostic purposes. Nevertheless, the size of the probes, usually 25–40 nucleotides, might limit their use to detect pathogens with high genome variability between isolates, where an identical sequence cannot be found without multiple mismatches. In this report, we describe a 5' nuclease assay, to detect Apple stem pitting virus, based on the use of a shorter probe which is chemically modified with a minor groove binder in order to increase duplex stability and raise the melting temperature to a value suitable for real-time analysis. The short size of the probe, which is critical to target a conserved cluster sequence of 14 nucleotides in the RNA polymerase gene, circumvents the genome variability of the virus. The assay correlates at 96 percent with gel analysis and is more reliable than biological indexing to detect Apple stem pitting virus field isolates. It is fast and fully compatible with automation, and therefore particularly suitable for plant certification. 相似文献
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