I would like to separate DNA-fragments (PCR-products) using HPLC. The crux is, that the fragments are all of the same length but differ slightly in the sequence.
My questions are:
- are there any protocols for this kind of separation
- what resolution is possible
- which columns should be used
- what conditions would be preferable
- any suggestions about this topic

Thank´s in advance

tomtom

Denaturing HPLC may be effective for fragments which differ in their terminal bases. See Anal. Biochem 212, p. 351 for examples. Anal. Chem 67, p. 578 also discusses the technique.

The separation of DNA fragments on a size basis is accomplished using an ion pairing reagent such as triethylammonium acetate, ACN solvent gradient and a nonporous polymeric column. However, you may be able to separate DNA on a size and sequence basis by making the DNA single stranded. This is accomplished by using less rigorous elution conditions (lower ACN concentrations) and raising the oven temperature to about 75 degrees C. Under these circumstances, single stranded DNA of the same length will have different selectivity for the column and can sometimes be separated even if they differ by only 1 base. The extent to which they can be separated depends on their length and the degree to which they differ. I've separated similar sequences up to about 100 bases.

One method to increase sequence dependence of the separation is to increase the polarity of the ion pair. But I haven't seen any successful work where the ethyl group of the ion pairing reagent is changed to 1 or more methyl groups.