Betaine significantly improves multiplex tetra-primer ARMS-PCR methods.

The tetra-primer ARMS-PCR method offers significant advantages over the commonly used methods to genotype single nucleotide polymorphisms. It offers fast and cost-effective detection and requires minimum level of expertise and basic instrumentation. The benefits of TP-ARMS-PCR increase exponentially upon multiplexing. However, several complications preclude the common use of multiplex TP-ARMS-PCR methods, primarily the lack of robustness and the difficulty of optimization. We have previously developed triplex and quadruplex TP-ARMS-PCR methods involving the simultaneous detection of up to three SNPs in a single reaction and utilized Betaine, a PCR additive used to enable amplification of GC-rich templates with strong secondary structures, in an attempt to facilitate method development and optimization. In the present communication, we introduced experimental data demonstrating the important effects of Betaine on our previous methods and its potential to overcome the ruggedness and robustness issues commonly found in TP-ARMS-PCR methods, and highlighted the general benefits of Betaine with respect to TP-ARMS-PCR. Our data support the routine inclusion of Betaine in all TP-ARMS-PCR methods, especially when multiplexing is concerned.