Daniel:Notebook/HiResChrPaint/2014-6-20
Probe Design (Started 06/17/2014)[edit]
Probe Mining[edit]
Well, it took a while, but the OligoArray script finished running and...
The Oligo.txt file has nothing in it. Most probes were denied due too high of a Tm. Guess I've got to redo the script but with bigger range for Tms. Sigh...
It would appear that I should have seen this coming. From a brief investigation into Tms of oligos, most oligos of length 60bp have Tms in the 70-100 range, which is way too high given the parameters I gave.
- Run OligoArray2
- java -Xmx4096m -jar /home/djacobse/blastdb_hg38/OligoArray2_1/OligoArray2.jar -i in_caskgene.fas -d hg19.fas -o oligo_caskgene.txt -r rejected_oligo.fas -R caskgene.log -n 30 -l 60 -L 60 -D 1000 -t 35 -T 95 -s 70 -x 70 -p 35 -P 80 -m "CCCC;GGGG;TTTTT;AAAAA" -g 2 &
FISH[edit]
I have done some reading while I am waiting for these OligoArray scripts to run, and I may need to increase the formamide concentration for my probes. It occurs to me that I have 60bp probes (genomic annealing), compared with the 32bp published by Beliveau et al. In general, the melting temperature (Tm) of short oligonucleotides increases with the length of nucleotides. Based on this, I took a look, and found this curve from IDT:
For probes of ~30bp (Beliveau), we see that the Tm is around 75 C. From running my analysis using OligoArray, I have noticed that my 60bp probes are closer to 80-90C, usually about 85C. So the additional 30 bp has added approximately 10C to the Tm. Why is this important?
Formamide is used during hybridization to lower the melting temp. From Roche guidelines on in situ hybridization (PDF here), they give a 0.72C decrease in melting temp of a probe per % formamide added. In terms of Beliveau, we see that
New Tm=Tm-0.72*50=75-36=39C
39C is right in the range of temperatures Beliveau et al used for hybridization (37 or 42 were their recommendations). Using my Tm instead:
New Tm=Tm-0.72*50=85-36=49C
That is too high for accurate hybridization! Using the target of ~40C as the adjusted Tm:
New Tm=Tm-0.72*x=85-0.72*x 40-85/(-0.72)=62.5% formamide
So I want 65-70% formamide to achieve Tms of 38.2-34.6C respectively. These will make the Tms much more amenable to the hybridization steps. I will continue to make the new oligos, but in the meantime this gives me some wetlab work to do. CERC just reopened, and Chris Stubban (CERC Manager) says the Biosafety Cabinets will be online Monday. Since I'll be at the Nature conference Monday/Tuesday, it will be Wednesday that I resume FISH experiments.