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Study for environmental monitoring to deduce genomic instability with mutation spectrum

利用突变谱推断基因组不稳定性的环境监测研究

基本信息

批准号：
18590133
负责人：
MASAMI Yamada
金额：
$ 2.57万
依托单位：
National Institute of Health Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590133/
关键词：
Mutation spectrum Spontaneous mutation Genome Archea Random mutation capture DNA

项目摘要

Mutation detection methods through phenotype alteration on the target genes always have a possibility to bias the results of the study. Moreover, they cannot be applicable to the mutations generated on un-expressed genes. Thus, it has been expected to find alternative methods to detect gene mutations without using phenotype changes, especially in sensitive, simple and easy procedures. Random mutation capture (RMC) method is one of the candidates, which measures mutation randomly generated on genome without any specific target genes.After digestion of bacterial genomic DNA with five kinds of restriction enzymes at 37 degree C for 16 hours, the DNA was purified with ethanol precipitation. About 400-bp probe DNA containing the target sequence, 5'-TCGA-3', was prepared by PCR using a 5'-biotynirated primer/un-biotinylated primer set with dUTP, dATP, dCTP and dGTP mixture. The probe was amplified with genomic DNA as template and purified with a microspin column. The biotinylated probe was b … More ound to streptavidin immobilized on magnetic beads for three hours at room temperature. The resultant probe was used as labeled magnetic probe. The digested genome fragments were hybridized with the labeled probe at 37 degree C for 16 hours, then double stranded DNA formed on the beads were captured with magnet and recovered. The double stranded DNAs formed with the target sequence and a labeled probe were treated with TaqI enzyme, which digests the target sequence TCGA, at 65 degree C for one hour, then denatured at 95 degree C for one minute and re-annealed at 50 degree C for three minutes to cut the DNA which has TaqI site with no mutations. After repeating this cycle five times, the sample was treated with uracil-DNA glycosylase to remove the probe which has no mutation at the TCGA site. Quantitative PCR was carried out at the end to measure the number of the target sequence recovered and the number of the fragment which has mutation at the TCGA site.Sensitivity of this method was not enough to detect spontaneous mutation frequency on bacterial genomic DNA. One big reason to be considered would be pseudo positive clones after incomplete digestion with Taq1 enzyme. The RMC method will be subjected into mitochondrial DNA. Less

通过靶基因的表型改变进行突变检测的方法总是有可能使研究结果产生偏差。此外，它们不能适用于在未表达基因上产生的突变。因此，人们期望找到替代方法来检测基因突变，而不使用表型变化，特别是在敏感，简单和容易的程序。随机突变捕获法（Random Mutation Capture，RMC）是一种检测细菌基因组上随机产生的突变而不需要任何特异性靶基因的方法，将细菌基因组DNA用5种限制性内切酶在37 ℃消化16小时后，用乙醇沉淀法纯化DNA。通过PCR使用5 '-生物素化引物/未生物素化引物组与dUTP、dATP、dCTP和dGTP混合物制备含有靶序列5'-TCGA-3 '的约400-bp探针DNA。以基因组DNA为模板扩增探针，用微量离心柱纯化。生物素化探针为B ...更多信息在室温下，与固定在磁珠上的链霉抗生物素蛋白接触3小时。所得探针用作标记的磁性探针。将消化的基因组片段与标记的探针在37 ℃杂交16小时，然后用磁体捕获并回收在珠上形成的双链DNA。将由靶序列和标记探针形成的双链DNA用TaqI酶（其使靶序列TCGA变性）在65 ℃下处理1小时，然后在95 ℃下变性1分钟，并在50 ℃下再退火3分钟，以切割具有无突变的TaqI位点的DNA。重复该循环5次后，用尿嘧啶-DNA糖基化酶处理样品以除去在TCGA位点没有突变的探针。最后用定量PCR检测回收的靶序列数和TCGA位点突变的片段数，该方法灵敏度不足以检测细菌基因组DNA的自发突变频率。考虑的一个重要原因是用Taq 1酶不完全消化后的假阳性克隆。RMC方法将用于线粒体DNA。少