High GC PCR: Difference between revisions
mNo edit summary |
mNo edit summary |
||
| Line 1: | Line 1: | ||
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. | 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: | <strong>Tips for successful high GC PCR:<strong/> | ||
Additives such as dimethyl sulfoxide (DMSO) can be added to reaction mixtures up to a final concentration of 1-10% can help denature DNA and promote specificity. | Additives such as dimethyl sulfoxide (DMSO) can be added to reaction mixtures up to a final concentration of 1-10% can help denature DNA and promote specificity. | ||
Revision as of 16:50, 3 July 2019
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) can be added to reaction mixtures up to a final concentration of 1-10% 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/).
Some specific polymerases can aid high GC DNA amplification. 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.
