Betaine Enhancer Solution 5 M is one of the most effective additives over a wide range of different templates, including GC-rich sequences and templates known to be extremely difficult to amplify. Betaine Enhancer Solution 5 M lowers the DNA melting temperature and has an enhancing effect on the polymerase.
The performance of Betaine Enhancer Solution 5 M is superior compared to standard enhancers such as formamide, DMSO, TMAC, BSA and non-ionic detergents. Betaine Enhancer Solution is an excellent enhancer especially when used with GC-rich regions or templates with a high degree of secondary structures.
In detail, Betaine binds preferentially to AT rich sequences in the major groove, thereby stabilizing AT rich regions of the DNA. Because AT forms 2 hydrogen bonds and GC forms 3, the bonding of AT is less stable than the one of GC. As a consequence of the stabilizing effect of Betaine on AT bonding, the stability of AT bonding and GC bonding is brought close to an equal level. At the same time, Betaine has a sequence independent destabilizing effect on all DNA. Summarized, the Tm of AT rich and GC rich sequences are equalized and the overall Tm is lowered. Furthermore, Betaine aids the processivity of thermostable polymerases and reduces "pauses" in polymerization caused by secondary structure that can induce the polymerase to disassociate from the DNA strand.
Betaine has a decreasing effect on the melting temperature of DNA and primers. Therefore, denaturation temperatures as well as primer annealing temperatures should be reduced by 1 – 5 °C. The optimal annealing temperature should be determined individually for each reaction.