The most significant function of DHPLC is mutation detection. Partially denaturing HPLC is used to detect unknown sequence variations. Heteroduplexes are generated before analysis. This step is essential for the detection of X-linked mutations in males and homozygous mutations. The screening throughput can be increased by mixing several test samples with 1 reference sample.
As has been mentioned above, the ideal size of PCR products is 150–450 bp for detection of unknown sequence variations. Long DNA fragments tend to have more than 1 melting domain and require several column temperatures for complete screening of the fragment.
There are occasions in which several sequence variations are found within a small DNA region in different chromosomes (i.e., in different individuals). These are well illustrated by the diverse mutations in the CFTR and HBB genes. Mutations in CFTR result in cystic fibrosis whereas mutations in HBB give rise to β-thalassemia or sickle cell disease. Distinct mutations in a PCR product usually give consistent distinct chromatograms.
Therefore, partially denaturing HPLC can also be used to genotype known sequence variations, particularly known mutations, once their corresponding distinct chromatograms have been established. However, it is still possible that different mutations may share indistinguishable chromatograms.
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