Bio 3125 Spring 2009 
Lecture Time and location to be announced

Lab Time and location to be announced

Instructor - Wayne Potts
office hours: TBA  (or by appointment)
585-9677 rm 582a ASB

TA - James Ruff
office hours: TBA (or by appointment)
585-9678 rm 580 ASB

Syllabus and additional resourses Student Papers 2009



This is a lab course. The goal is to learn molecular genetic techniques and computer analysis techniques (bioinformatics) that are used to test hypotheses about molecular evolution or test hypotheses about population-level phenomena. In this course I have attempted to keep the theoretical details of molecular evolution basic and simple. There is an entire molecular evolution lecture course that deals with these theoretical aspects in detail. This lab and bioinformatics course is largely based on the proposition that you learn by doing. You will learn basic molecular genetic techniques by doing a series of projects, often based on various analyses of your own DNA. Computer-based analysis tools will be learned through a series of homework exercises and demonstrations. The projects represent interesting problems involving molecular data that are current areas of scientific investigation. An overview of the techniques, analysis tools and projects follow.


  • Pipetting
  • Centrifugation
  • Reagent/buffer/media preparation
  • DNA extraction
  • Restriction digestion and restriction mapping
  • Agarose and acrylamide gel electrophoresis
  • Polymerase chain reaction (PCR)
  • Microbial (aseptic) culture techniques
  • Cloning in microbial plasmid vectors
  • Plasmid preparations
  • Sequencing (template preparation for automated sequencing)
  • Microsatellite genotyping for parentage and forensic analysis

  • Pubmed – Scientific literature database
  • Genbank – database of DNA and protein sequences
  • BLAST – a sequence similarity searching tool
  • Restriction Site Analysis – a restriction endonuclease analysis program
  • PRIMER - PCR primer design
  • Sequencher – a sequence manipulation and evaluation program
  • Clustal – a multiple sequence alignment program
  • PAUP – a program for constructing phylogenetic trees
  • MacClade – a program for analyzing character evolution and manipulating and evaluating phylogenetic trees
  • KINSHIP – a program for evaluating relatedness from genetic data
  • SNAP – a program to test for diversifying selection using a synonymous/non-synonymous substitution analysis


    Restriction Digests of Lambda Phage

    Restriction endonuclease enzymes cut DNA at specific DNA sequence sites. They generally function as a bacterial immune system by degrading viral genomes. Their discovery was critical to the development of recombinant DNA technology, because they were the first tool that allowed DNA to be cut at specific sites. Two restriction enzymes will be used to cut Lambda phage DNA. The entire genome of Lambda will be downloaded from Genbank and computer-based restriction analysis for the two restriction enzymes will be conducted. The resulting restriction fragments will be used as size markers on agarose and acrylamide gels throughout the rest of the course.

    Experimental Evolution of Streptomycin Resistance and Compensatory Mutations

    Evolution is usually thought to occur so slowly that it can only be studied indirectly. Over the past decade this notion has proven false as it has been shown that evolution can occur rapidly enough to be studied in real time experiments. We will study the experimental evolution of antibiotic (streptomycin) resistance in the bacteria Salmonella. We will also study the troubling observation that resistant bacteria can evolve compensatory mutations that remove the initial cost of resistance, making antibiotic resistance stable in the pathogen. The evolution of antibiotic resistance is a major problem for humans as antibiotic resistance is evolving in human pathogens at a much higher rate than the discovery of new antibiotics.

    My DNA Extraction

    You will each extract DNA from your own cells collected from cheek scrapings. These genomic DNAs will be used in the following two projects.

    Cloning and Sequencing MHC DQα1 Genes and Mitochondria

    Everyone will PCR amplify their own MHC (major histocompatibility complex) DQα1 gene and a segment of the mitochondrial D loop. The PCR products from mitochondria will be directly sequenced and those for MHC will be cloned into the plasmid pUC. (Note: Cloning technology is arguably the other major advance (after restriction enzymes) that allowed the molecular genetic revolution to take off in the 1970s). These plasmids will be prepared for sequencing. The resulting sequence information will be distributed to all class members who will then construct phylogenetic trees, including sequences from great apes, neanderthals and humans downloaded from Genbank. The phylogenetic trees for the MHC and mitochondrial genes will differ dramatically and you might find the results profound. The MHC sequences will also be analyzed for indications of diversifying selection.

    Genetic Fingerprinting with Microsatellite Loci: CSI Bio3125

    There are tens of thousands of microsatellite loci scattered throughout the genome. Microsatellite loci contain a tandemly repreated di, tri or tetra nucleotide motif. For example, the sequence gcactctctctctctctgaatc is a ct dinucleotide microsatellite with 7 repeats. These loci have a high mutation rate where the number of tandemly repeated units change. Consequently, they are highly polymorphic and thus become useful genetic markers that have revolutionized both population studies and forensics. For example, about 25 years ago we believed most avian species were monogamous, because socially they acted monogamous. We now know using genetic markers that the opposite is true; about 90% are polygynous or polyandrous. The now well known example from forensics is that many criminals are convicted largely on DNA evidence, but more importantly, many accused/convicted innocent people have been exonerated based on DNA evidence. It has revealed how inaccurate the justice system has been.

    Using genomic DNA from your cheek DNA extractions (coded) and from one or more planted families, you will microsatellite genotype all individuals and apply the data to the following problems: Parentage / relatedness study - You suspect there are one or more families (parents and offspring) among a group of individuals, such as might occur in a wolf pack or post-breeding swallow assemblage. You will use your combined microsatellite data to identify the first degree relatives by comparing the proportion of allele sharing between pairs of individuals. This will be accomplished both by inspection and by using the RELATEDNESS program by Goodnight and Quellar. You will then attempt to determine who the parents and kids are from the genetic data?

    We will also conduct a forensic study where we attempt to identify the murderer/rapist from a tissue sample taken from the crime scene. We will calculate the probability that there is another individual with this genotype in the population defined by genotypes found in the class.