High GC PCR
High GC PCR
Actinobacteria typically have a high GC content in their DNA. High GC DNA templates can be difficult to amplify by PCR because of stronger hydrogen bonding and possible formation of secondary structures.
Tips for successful high GC PCR
- Additives such as dimethyl sulfoxide (DMSO) or betaine can be added to reaction mixtures up to a final concentration of 1-10% or 0.5 M, respectively, which can help denature DNA and promote specificity.
- When designing primers, assess properties such as melting temperature and the likelihood of secondary structure formation. A useful tool for this is Net Primer (http://www.premierbiosoft.com/netprimer/).
- When amplifying a region of DNA, try to find small regions of higher AT content that could form part of the primer sequence. This well help lower the Tm of the primers and reduce self-annealing and secondary structure formation. Try to aim for a primer with no more than 75% GC content.
- Some specific polymerases can aid high GC DNA amplification. High fidelity polymerases will help improve specificity.
- Q5® High-Fidelity DNA Polymerase comes with a high GC enhancer that can be added to reaction mixtures up to a final concentration of 10-20% to improve specificity.
General protocol for Streptomyces colony PCR
- Streak your strain of interest for single colonies on DNA medium.
- Pick a single colony and crush in 100µl 50% DMSO.
- Heat at 65ºC for ~20 minutes.
- Add 1µl of this to 10µl of PCR mix