The mode of operation is completely denaturing if the column temperature is maintained between 70°C and 80°C. Under such a high temperature, DNA molecules are completely denatured and become single-stranded. Completely denaturing HPLC can differentiate single-stranded (ss) DNA (and RNA) molecules with the separation depending on both the length and the base composition of the single-stranded nucleic acid molecules.
It can be used to analyze and isolate synthetic oligonucleotides and RNA molecules. More commonly, it is used to analyze the products from primer extension (PE) reactions (also known as minisequencing). In PE reaction, an extension primer is annealed immediately upstream of the polymorphic site.
In the presence of a modified DNA polymerase (e.g., Thermo Sequenase from GE Healthcare) and appropriate unlabeled dideoxynucleotides (ddNTPs), the primer is then extended in a template-dependent manner. The allele-specific extension products with different sequence compositions are then detected and discriminated by completely denaturing HPLC.
As such, completely denaturing HPLC provides a robust platform of medium throughput for genotyping known mutations or SNPs. The throughput can be increased by multiplexing several primer extensions in a single reaction.