Daniel:Notebook/HiResChrPaint/2013-11-13
Positive Controls for FISH[edit]
After a long hiatus for NSF and BWI, I'm now ready to start again. After talking with Dr. Zhang, we have two ideas to try for positive controls for the FISH experiment.
Positive Control 1: Alu Elements[edit]
The first uses ALU elements, which are repetitive sequences found throughout the human genome around 300 bp long. I've attached a concensus sequence I've been working with from Jurka and Smith (PNAS 1988):
File:ALU sequence JurkaSmithPNAS1988.png
I am going to amplify these elements from human DNA, attach fluorophores to them, and hybridize the amplified probes back to human DNA in cells. ALU should provide strong signal in many places, providing a good positive control for working FISH probes.
Positive Control 2: Single Color Amplification[edit]
If the problem with the multi-color FISH is low signal, we can amplify the signal by using single-color FISH. That is, amplify all four probe sets and couple them to a single fluorophore. Assuming the probes do in fact hybridize to DNA, the signal should increase considerably with single color FISH. My plan is to use Alexa 488, since that is (a) one of the most stable fluorophores I've used and (b) does not interfere with DRAQ5, the the dye that I can use to generically stain nucleii instead of DAPI. I'm using DRAQ5 because its excitation is within the visible spectrum, instead of DAPI, which is excited by UV (which the confocal does not have).