In my last post I mentioned the process of gene amplification through Polymerase Chain Reaction (PCR). I thought I might expand on the topic, since most of my week has revolved around diluting primers and combining them with other reagents to amplify specific sequences of DNA.
Some vocab that might help to read this post:
- Polymerase Chain Reaction (PCR): a molecular biology technique used to amplify a piece of DNA over and over again, allowing analysis of that gene
- Primer: a short complimentary nucleotide sequence created to bind a specific gene
- Locus: the specific location of a gene’s DNA sequence on a chromosome (plural: loci)
- Genome: the complete set of genes present in an organism
Again, the big picture: our project goal is to establish the magnitude of relation between six populations of E. Kennicotti in the Upper Cumberland, Laurel River, Ohio-Clarks, Green, Lower Tennessee, and Upper Tennessee.
Populations of E. Kennicotti have already been compared by morphology (the physical structures of organisms, ex: scale count), nuclear loci (genetic material located in the nucleus), and mitochondrial loci (genetic material located in the mitochondria, an organelle inherited from an individual’s mother). We are taking the project one step further by comparing ~25 microsatellite loci.
Though I talked about microsatellites briefly in my second blog post, here’s a refresher: microsatellites are nucleotide repeats found in an individual’s genome. The repeats don’t code for anything – while some DNA sequences make proteins, others like microsatellites act as glorified placeholders. (There are various theories on what they actually do.) The number of microsatellite sequence repeats differs between individuals. We can estimate the amount of genetic diversity between populations by establishing the variation in number of repeats.
Okay. The stage is set. So how do we visualize the number of microsatellite repeats across 25 loci in an individual? PCR! Wahoo!
We combine various reagents, including DNA polymerase, free nucleotides, and primers. We put this mixture into individual tubes, add DNA from different individuals to each, then stick the tubes in a thermal cycler. That program allows the DNA to:
- Denature. The strands of DNA are usually in a double helix, but in high temperatures the helix “unzips.”
- Anneal to the primer. Primers have been specifically constructed to bind to these DNA sequences. A forward primer binds to one of the unzipped strands, and a reverse primer binds to the other.
- Elongate. The DNA polymerase creates a new DNA strand complementary to the template strands in step 2.
Steps 1-3 repeat on a cycle to amplify the DNA. Image from wikipedia.
To visualize the product, we perform gel electrophoresis. But I’ll save that for another time.