Synthesizing probes

To run ISH, we need to synthesize probes that will tell us when our DNA sequence of interest has bound to the DNA on slides of our samples, indicating that the pathogen gene (can we say pathogene for short?) is present in the sea star tissues we fixed during the histology process. In the past, probes were only labeled on the end of the PCR-amplified segment, meaning that you needed a lot more of the target DNA present in the sample to have something show up in great enough quantities to visually observe. Now, probes are labeled within the PCR segment, so you can get a signal from much smaller amounts of DNA present.

To make our probe, we followed the procedure for normal PCR gene amplification, except that instead of plain nucleotides, we had some DIG mixed in with our nucleotides, so they would bind with our target DNA as it was synthesized. We needed to make sure that there weren’t already things that caused the same visual appearance as our probes, so we made a batch of controls that had no probes (using just plain nucleotides). If we are successful, the probe will show up when we drop it on the tissue slides and the control will not show up.

We also prepped for deparaffinizing our tissue slides. The paraffin keeps them preserved and stiff so we can look at them whenever we want, but this requires dehydrating all the cells and blocks access to the DNA. To do this, we need to take out the paraffin with xylene, then rehydrate the tissues with a mix of ethanol and water, slowly upping the water content. This process takes over an hour, but it will give us access to the DNA in the tissues so we can drop on the probes and see if they bind.

 

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