The PE reaction in combination with completely denaturing HPLC analysis offers a simple, robust and automatic genotyping platform because a single analytic condition can be used. The accuracy and sensitivity can be increased by using fluorescent-labeling method. Equipped with the DNASep-High Throughput (HT) column and the High Sensitive Detector, the 4500HT-HS model of the WAVE System (Transgenomic) is developed for high-throughput genotyping.
The PE/DHPLC genotyping platform has been used in the genotyping of the mutations of the hemochromatosis gene and the β-globin gene for the diagnosis of hereditary hemochromatosis and β-thalassemia respectively. Two mutation sites (C282Y and H63D) of the hemochromatosis gene were simultaneously genotyped in a multiplex format including the PCR, PE reactions and DHPLC detection.
For β-thalassemia, two different studies demonstrated the successful simultaneous genotyping of five common mutations within the β-globin gene. Amplicons containing the five common mutations were amplified, and the mutations distinguished by multiplex PE reactions followed by DHPLC analysis.
This approach would help the development of diagnostic mutation panels for the diagnosis of β-thalassemia and other genetic diseases showing extensive allelic heterogeneity.
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