Hello readers! Given that much of my upcoming research will involve the analysis of DNA sequences extracted from unidentified ribbon worms, I’m devoting this blog post to describing the procedures involved in procuring a specific gene of interest from a live specimen. Bear with me—this is an information-packed post—and enjoy my hand-drawn diagram of the processes involved (see below).
The procedure for procuring a gene of interest begins with tissue extraction from the specimen. We obtained tissue by snipping off the tip of a live ribbon worm with a razor blade (don’t worry, the tail will grow back!). Next, we ground the tissue in a small tube with a pestle, and added detergents to break open the individual cells and liberate the carefully-packaged DNA. We then spun this mixture at a high speed until the cell components (cell membrane, organelles, DNA) had separated based on particle size and density, allowing us to extract the DNA from the uppermost layer (see steps 1-2 in the diagram below).
With the DNA successfully extracted from the tissue, the next goal was to make copious copies of the gene of interest so as to create an extensive stock of the isolated gene to send for sequencing. Gene amplification is performed via PCR (polymerase chain reaction), where enzymes specific to the focal gene target and initiate the repeated copying of the gene of interest. The result of PCR is an abundance of copies of the single, isolated gene; this result is then verified via gel electrophoresis to ensure that the collection of gene copies is uncontaminated (it is easy to sneeze and accidentally amplify your own DNA!). Steps 3-4 in the diagram below illustrate the process of PCR gene amplification.
Gel electrophoresis serves as a means of separating DNA fragments on the basis of size and charge. A small amount of the PCR product (copies of the isolated gene) is placed on an agarose gel which is then treated with an electrical current. The DNA migrates towards the positively-charged end of the gel; small DNA fragments migrate most quickly through the pores of the gel and create a gradient of fragment on the basis of size along the length of the gel. The resulting gradient can be visualized under UV light. For PCR product, the goal is to produce a single bright band (meaning that the DNA fragments are the same length and are therefore the single isolated product) rather than a gradient of assorted fragment lengths (see step 5 in the diagram below).
Once gel electrophoresis has verified that PCR has amplified a single gene, the gene product is further purified, quantified, then shipped off to a company for sequencing. I will devote future blog entries to the exciting processes involved in determining specimen identity based on gene sequences, and will leave you this week with pictures of the sea star and sea urchin larvae we fertilized in past weeks (see below).
Patiria miniata sea star larva (top) and Strongylocentrotus purpuratus sea urchin larva (bottom), imaged under a compound microscope.
“Instructions for living a life./ Pay attention./ Be astonished./ Tell about it.” (Mary Oliver)