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

点击翻译按钮获取中文摘要

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.