Thermoregulatory Tracing Part I: DNA Synthesis and Purification

In situ hybridization (ISH) is a laboratory protocol that takes advantage of nucleic acid base pairing to identify the messenger RNAs present in cells.  Essentially the experimenter makes an RNA probe that is complimentary to the RNA of interest that will produce a color reaction in the cells where the RNA of interest is present.  This protocol is extremely useful because based on the central dogma (DNA –> RNA –> protein), scientists can use it to examine gene and protein regulation in cells.

The first step in an ISH protocol is finding or building a plasmid that contains the gene of interest.  A plasmid is a small circular loop of DNA that is found in bacterial species.  Plasmids are important because they are easy to insert new genes into and they allow for essentially unlimited gene cloning (thanks to the speedy replications of bacteria).  In my case, I contacted Dr. Salah El Mestikawy, who worked extensively with the VGLUT2 protein in France and Canada.  Dr. El Mestikawy was kind enough to send me purified plasmid DNA that contained the gene I was interested in.

Once I received the DNA, the next step was to transform bacteria with it for cloning.  Bacterial transformation is a process in which bacteria can take up plasmids and incorporate them.  Once the plasmid is incorporated into the bacteria, they multiply along with the plasmid of interest inside them, thus cloning the DNA.  In the lab, we use specially designed bacteria that are more likely to take up the plasmid and replicate with it.  To cause transformation to take place, the bacteria are put under stress by keeping them at very low temperatures and then heat shocking them for a short period, which essentially causes the cell membranes to become permeable and allows for the uptake of the plasmids.  There are several checks in place as well to ensure that the bacteria growing are the ones we are interested in.  First, the plasmids we are using contain a gene that confers antibiotic resistance to the bacteria it is in.  By putting the bacteria in growth media that contains the antibiotic, scientists can be sure that the bacteria that are growing contain the plasmid.  The other checkpoint is the fact that the gene insertions are placed in such a way in the plasmid so that the a gene that codes for B-galactosidase is cleaved.  As a result, the bacteria will not be able to break down a molecule known as X-gal, which is also placed in the growth medium.  Bacterial colonies that can break down B-galactosidase appear blue, whereas colonies that cannot break it down will appear white.  Thus, scientists know that the bacteria has the gene of interest inserted in the plasmid if the colony appears white.

The next step in the process is DNA purification.  There are several kits that are pre-made specifically for DNA purification purposes.  These kits use several important aspects of DNA in order to separate it from the rest of the molecules in the cell.  During the process, the cells are lysed (broken open) and centrifugation is used to separate out the cellular debris.  After that, the kit takes advantage of the various properties that DNA has (largely its solubility in certain solvents) to purify it.

Once the DNA purification is completed, the experimenter can utilize a restriction digest procedure to ensure that the DNA is correct.  Restriction enzymes are enzymes developed by bacteria to fight viral infections.  These enzymes cleave DNA at known sites based on the base pair sequence.  By knowing where these restriction enzymes cut the DNA, scientists can use them to cut out certain regions of the DNA.  This procedure is used after DNA purification to cut the DNA on either side of the gene of interest so that it is separated from the plasmid.  The DNA is then run on an agarose gel (which separates DNA sequences by size) to see how long the sequences are and to see whether they match up with what would be expected for the plasmid of interest.