Development of molecular probes for genome-wide genotyping by restriction landmark genomic scanning (RLGS) method.

通过限制性标志基因组扫描（RLGS）方法开发用于全基因组基因分型的分子探针。

基本信息

批准号：
08556047
负责人：
SHIOTA Kunio
金额：
$ 9.34万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

Recent advance in the artificial insemination technique by using frozen-preserved semen greatly enhanced a production of economically superior livestock animals. It, however, ironically helped cryptic genetic disorders to spread world wide in an unexpected speed. Thus, it has been needed to establish effective way of genome-wide screening for genetic abnormalities in livestock animals. To this end, it was aimed to develop a system to identify changes in a structure of genomic DNA by a restriction landmark genomic scanning (RLGS) method. In the RLGS, genomic DNA is digested by two or more different kind of restriction enzymes, and separated by the 2-dimensional gel electrophoresis (2D/E). It is, therefore, possible to sensitively identify global genomic changes such as point mutations at the recognition sites of restriction enzymes, deletions, translocations, etc. RLGS should be also useful to establish a linkages between a certain valuable phenotype and a set of multiple genes.In this study, we analyzed CpG islands of rat genomic DNA by RLGS using NotI, a methylation sensitive enzyme. As a result, we found approximately 1% of the "RLGS spots" show tissue-specific distribution, suggesting that these spots are tissue-specifically methylated/demethylated. Of these spots, we could subsequently clone several genomic fragments which appeared to be demerhylated in placenta but not in kidney. Sequence analysis of isolated clones revealed that one of the clones encodes mitochondrial C3 transporter gene which appeared to be relatively highly expressed in the placenta but low in kidney in further analysis. These results showed that the RLGS is an effective way of identification and isolation of genomic DNA fragments by genome-wide scanning for the genetic changes.

通过使用冷冻保存的精液大大增强了经济上最优质的牲畜动物的产生，该技术的最新进展大大增强了。然而，具有讽刺意味的是，它有助于神秘的遗传疾病以出乎意料的速度传播全球。因此，需要建立对牲畜遗传异常的全基因组筛查的有效方法。为此，它的目的是开发一个系统，通过限制性地标基因组扫描（RLGS）方法来识别基因组DNA结构的变化。在RLG中，基因组DNA被两种或多种不同类型的限制酶消化，并通过二维凝胶电泳（2D/e）分离。因此，有可能敏感地识别全球基因组变化，例如在限制酶，缺失，易位等识别位点的点突变。RLGS也应该有助于建立一定有价值的表型和一组多个基因之间的链接。在这项研究中，我们分析了使用RLG的CPG岛，使用RLGS noti noti noti noti noti。结果，我们发现大约1％的“ RLGS”显示了组织特异性分布，这表明这些斑点是组织特异性甲基化/脱甲基化的。在这些斑点中，我们随后可以克隆几个基因组片段，这些基因组片段似乎在胎盘中被脱粒，但在肾脏中却没有。分离的克隆的序列分析表明，其中一个克隆编码线粒体C3转运蛋白基因，该基因在胎盘中似乎相对高度表达，但在肾脏中较低。这些结果表明，RLGS是通过基因组范围扫描遗传变化来鉴定和分离基因组DNA片段的有效方法。