Restriction Mapping and Sequencing of Mouse Mitochondrial DNA
Koh, Jeffrey L.
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I was involved in developing a restriction enzyme site map of this particular area, and beginning to sequence it. Work on restriction sites in mouse mitochondrial DNA has been done before (Moore et. all 1977), but no useful sites had been found in this area. The general strategy involves first cloning the desired DNA fragment into a plasmid from Escherichia coli, which can then be used as a source of DNA. Second, since fragments of only about 250 nucleotides in size can be adequately sequenced, sites where enzymes cut must be found close to or even within the gene, so that the sequencing can be done. Once an adequate restriction map has been developed, sequencing is done from on the sites into the gene. Confirmation can be done by sequencing from other sites on the other side of the gene or within the gene. By looking at the sequence, perhaps through a computer program, the actual gene sequence can be determined by using knowledge of initiation sequences, termination sequences, and possibly the known sequence of the same gene from another animal. In this research, useful restriction sites were found and sequencing was begUn, but time did not allow for the determination of the actual sequence of the gene.