Isolation of Caenorhabditis elegans genomic DNA and detection of deletions in the unc-93 gene using PCR

Abstract

PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the nematode Caenorhabditis elegans and use PCR to detect deletions in the C. elegans unc-93 gene. In advance of the exercises, wild-type and three different unc-B3 deletion mutant strains are grown, harvested, and frozen by the instructor. In one approach, students isolate genomic DNA from each strain using a genomic DNA isolation kit and use agarose gel electrophoresis to analyze the DNA and to estimate its concentration. PCRs using primers directed to two different regions of the unc-93 gene are carried out on the genomic DNA from wild-type and mutant strains, and the PCR products are analyzed by agarose gel electrophoresis. Students analyze the gel to determine the approximate location and size of deletions in the three mutant strains. Alternatively, students may lyse single nematodes and carry out PCR in one laboratory session. These exercises should be easily adaptable to detection of well characterized deletions in any organism. © 2005 by The International Union of Biochemistry and Molecular Biology.