I am trying to use qPCR to genotype single nucleotide polymorphisms (SNP). Some of the SNPs are located in GC poor regions and I cannot get a probe Tm 8-10 degrees higher than my primer Tm. Do you have any suggestions on how I can increase my Tm without increasing the probe length above 30bp? Thanks!
You could try adding locked nucleic acid residues into your probe, which increases the Tm. There are a few restrictions of where you can place them, and not every provider can produce them (for example, IDT can but Biosearch can't), but it's a very simple way to increase your Tm without having to change target region or redesign your primer/probe.
Article Locked nucleic acids in PCR primers increase sensitivity and...
You could try adding locked nucleic acid residues into your probe, which increases the Tm. There are a few restrictions of where you can place them, and not every provider can produce them (for example, IDT can but Biosearch can't), but it's a very simple way to increase your Tm without having to change target region or redesign your primer/probe.
Article Locked nucleic acids in PCR primers increase sensitivity and...
Exiqon is another provider of LNA custom oligos if you're wanting to get some pricing information to compare to LifeTech and IDT.
+1 regarding LNAs. Exiqon is the patent holder of LNAs and the others license their products. I think IDT no longer sells custom LNAs but instead only their pre-designed PrimeTime probes:
http://www2.idtdna.com/pages/products/genotyping/lna-primetime-probes/lna-notification
You can also play around with different additives in your PCR:
http://bitesizebio.com/24/pcr-problems-try-an-additive/
Good luck!
You can use oligopyrrolecarboxamides MGB probe.
https://www.researchgate.net/publication/5677135_TaqMan_probes_based_on_oligonucleotide-hairpin_minor_groove_ligand_conjugates
Article [TaqMan probes based on oligonucleotide-hairpin minor groove...
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