Woodrat Hunting, Part 3
Posted On: 03/05/2018 - 11:57am, Posted By: Heather Farrington, Curator of Zoology
To catch up on the background of our Allegheny Woodrat project, check out part 1 and part 2 of this blog post series.
The woodrat project has now moved into the DNA lab at CMC’s Geier Collections and Research Center! The first step is to extract the DNA from the tissue samples collected from our woodrats. DNA is released from the cells by “digesting” the tissue in a special type of detergent (to break down the fatty cell membranes) with an enzyme called Proteinase K (to break down proteins in the cells) at high temperature. Through a series of wash steps, the DNA is separated from the broken-down components of the cells, purified, and concentrated. What we end up with is just a few drops of clear liquid containing strands of DNA – it doesn’t look like much, but it goes a long way in the lab!
We don't need much tissue to obtain DNA. Here we show the ear punch sample collected from one of our woodrats next to a dime for size comparison. That little bit of tissue is enough to run hundreds of genetic analyses!
Examining the whole genome of an organism at once is a daunting task – there’s just too much information. To simplify things, we only look at specific, small chunks of the genome. These pieces of DNA are isolated and copied thousands of times using a process called Polymerase Chain Reaction (PCR). The PCR products are then run on our DNA sequencer. Depending on the research question, some scientists want to know the actual sequence of A, G, C, and T (nucleic acid base pairs Adenine, Guanine, Cytosine and Thymine) in the genetic code, others just want to know the length of the DNA fragments produced by the PCR reaction. The woodrat project uses the latter – simple repeat units (for example, ACACACAC or TGTGTGTG) in the DNA called “microsatellites” make the DNA fragments vary in size depending on the number of repeats present.
Woodrats have a very similar genetic structure to ours – they are “diploid” organisms, meaning they have two copies of each chromosome (one inherited from mom and one from dad). Chromosomes are made up of many genes. Sometimes the two inherited copies of a gene match (known as homozygous) and sometimes they don’t (known as heterozygous). Different versions of the same gene are called alleles. For each woodrat, we will identify what alleles are present in 11 different microsatellite fragments to generate a genetic profile for each individual.
The picture below shows what the raw data look like coming off the DNA sequencer. Each horizontal bar represents one woodrat sample. The yellow, blue, and green peaks represent three different microsatellites, or areas of repetition. The first individual at the top is heterozygous for the yellow and green microsatellites, but homozygous for the blue. The lengths of DNA fragments can be determined by comparing them to the red peaks of the “size standard”.
Here is a screen shot of what we see while the DNA sequencer is running. These "traces" are uploaded to a special computer program that isolates each color in the trace and tells us the exact size of the DNA fragments.
Once we identify all the alleles for each individual, we hope to discover how closely related woodrats in a given area are, how far they disperse (move) when they leave the nest, whether animals occupying different rocky outcrops form isolated populations, and if our populations are small enough to suffer from inbreeding depression. We will also establish baseline genetic diversity levels in each population for continued monitoring efforts in the future.
Stay tuned for the exciting conclusion of our woodrat project blog series! What will the data tell us about these elusive and adorable animals? I can’t wait to find out myself! Back to the lab!