After sample injection, the signal intensity of DHPLC profile is shown real-time on the computer screen. A DNA fragment appears as a peak in the chromatogram with the corresponding intensity and retention time. Interpretation of DHPLC data is based on the comparison between sample and reference chromatograms.
For sizing of a DNA fragment, the sample peak is compared with a series of DNA fragments of known size or commercial size standards. Because the retention time increases with increasing DNA fragment size, the retention time becomes a measure of DNA fragment size. Accordingly, the size of a DNA fragment is determined by comparing the retention time obtained and the retention times of the size standards. In mutation detection, the elution profile of a test sample is compared with that of the wild type sample.
Any altered elution profile such as shoulder peak and additional peak(s) implies the presence of heteroduplexes and hence mutation(s) in the DNA fragment. The DHPLC profile of a test sample showing any aberrant elution profile is then confirmed by direct sequencing. In allelic discrimination, the alleles are distinguished by the retention times of allele-specific PE products.
Different sequence compositions of PE products give different retention times, which can then be verified by DNA samples of known genotypes. The genotype of a test sample can be determined on the basis of the number of elution peaks and their corresponding retention times. In addition, the detected DNA samples can be quantified by the peak height or area. Accordingly, the amount of DNA samples and the allele frequencies of DNA pools can be estimated.