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3. 发明申请

WO2007105041A2 A METHOD FOR THE SCREENING OF BACTERIAL ISOLATES 审中-公开
标题翻译：一种筛选细菌分离物的方法
公开(公告)号：WO2007105041A2
公开(公告)日：2007-09-20
申请号：PCT/IB2007000136
申请日：2007-01-22
申请人： COUNCIL SCIENT IND RES , PUROHIT HEMANT JYOTISWARUP , KAPLEY ATYA , RAJE DHANANJAY VASANT , DEVOTTA SUKUMAR
发明人： PUROHIT HEMANT JYOTISWARUP , KAPLEY ATYA , RAJE DHANANJAY VASANT , DEVOTTA SUKUMAR
IPC分类号： C12Q1/68
CPC分类号： C12Q1/689
摘要： Present invention relates to a method to determine the genotype of organisms by RAPD analysis and more specifically, to establish the relatedness of individual organisms across and within species. RAPD uses genotypic information of an organism to give an organism specific DNA fragment of different sizes. The present invention provide methods and a set of oligonucleotide primers for performing amplification and other enzymatic reactions on nucleic acid molecules that have been collected directly as environmental DNA or DNA derived form pure isolates. More specifically, the present invention relates to a novel method of genetic analysis using a set of sub-sequence, which occurs as inverted repeats in different genome with different frequencies. All bacterial cultures used in this study have been isolated from activated biomass collected from effluent treatment plants. The bacteria have been sub-cultured repeatedly to obtain pure cultures. All plating has been carried out on Luria Broth plates with 2% agar. The 16S rRNA gene has been amplified using universal primers to confirm the eubacterial nature of the isolates. The primers used to amplify a 1466-bp product were 27F forward primer 5'- AGAGTTTGATCMTGGCTCAG-3' and 1492 reverse primer 5'- TACGGYTACCTTGTTACGACTT-Hence, in defined conditions two genome samples could be differentiated from each other. These features are applicable to DNA fingerprinting, marker assisted selection, genotyping, and high throughput laboratory screening methods for culturable microbes from any environmental niche.
摘要翻译：本发明涉及通过RAPD分析确定生物体基因型的方法，更具体地说，用于建立物种间和物种内个体生物体的相关性。 RAPD使用生物体的基因型信息给出不同大小的生物体特异性DNA片段。本发明提供了用于对已经直接作为环境DNA或源自纯分离物的DNA收集的核酸分子进行扩增和其他酶促反应的方法和一组寡核苷酸引物。更具体地，本发明涉及一种使用一组子序列的遗传分析的新方法，其以不同基因组的不同频率的反向重复出现。本研究中使用的所有细菌培养物都是从污水处理厂收集的活性生物质中分离出来的。细菌已经被重复培养以获得纯培养物。在含有2％琼脂的Luria肉汤培养板上进行所有的电镀。使用通用引物扩增16S rRNA基因以确认分离株的真细菌性质。用于扩增1466bp产物的引物是27F正向引物5'-AGAGTTTGATCMTGGCTCAG-3'和1492反向引物5'-TACGGYTACCTTGTTACGACTT-因此，在确定的条件下，两种基因组样品可以相互区分。这些功能适用于DNA指纹识别，标记辅助选择，基因分型和高通量实验室筛选方法，可用于任何环境生态位的可培养微生物。

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