A Research-based laboratory course in Molecular Biology, Genetics, and Evolution
Author: Eric Spana
Affiliation: Duke University
Keywords: f. classroom undergraduate research experience (CURE)
A introductory level course at Duke University in Molecular Biology, Genetics, and Evolution required a lab component that spanned as much of these three subjects as possible. I have developed a lab that uses local, wild-caught fruit fly species (mostly Drosophila) as the starting point for a laboratory portion in the course. Students choose flies from the collection and use PCR and sequencing of the Cytochrome Oxidase I gene to identify the species via DNA barcoding analysis. After phylogenetic analysis of the collected species, students amplify a region of a second gene, the Tyrosine Hydroxylase gene (TH) using degenerate PCR primers. This PCR product is then cloned into a TA cloning vector. After transformation, mini-preps, and restriction digest analysis, students can identify which samples contain the appropriate cloned DNA. Sequencing and analysis of the plasmid insertions follows and a number of evolutionary analyses are performed on the sequence. The region of TH that is being sequenced covers both introns and exons, and there is a cis-regulatory element in one intron that drives expression in the larval brain in D. melanogaster. Alignment of the TH region sequences identified a few highly conserved regions within the intron that contains the cis-regulatory element. I have run this course at two different Universities so far: Duke University and Duke Kunshan University, and have identified ~22 species in Durham, NC and ~11 in Kunshan, China with an overlap of only three species and at least one unidentified species in each location. This laboratory gives students experience in many common molecular biology techniques: PCR, cloning, plasmid DNA isolation, restriction digests, agarose gels, and sequencing as well as analysis of their own data for gene structure, reading frames, codon usage, dN/dS ratio, MK test, phylogeny. An online version of this lab used extensive BLAST and gene annotation from 80+ sequenced genomes to arrive at a similar result.