Clinical Diagnostics and Research tools
Gap PCR techniques (amplification using oligo-primers flanking deletion breakpoints) are used to detect common alpha thalassemia deletion mutations, alpha gene duplication, and other globin gene deletions, such as Hb Lepore and HPFH.
Gap PCR is based upon the inability of PCR primers complementary to DNA sequences that are far apart to direct amplification unless a deletion brings them closer together. PCR primer pairs are designed to flank a known deletion, generating a unique amplicon that will be smaller in the mutant sequence compared with the wild type. The presence or absence of PCR product is detected by electrophoresis. Primers specific for 7 of the most common alpha thalassemia deletions, as well as the CS point mutation are multiplexed to detect the mutations most often responsible for HbH disease. This technique is also used to detect the delta/beta globin gene crossover responsible for Hb Lepore and the large deletions responsible for hereditary persistence of fetal hemoglobin.
Alpha thalassemia deletions (and CS)
Alpha gene duplications
Delta-beta deletions (HPFH-1, HPFH-2, HPFH-3, HPFH-7, Lepore)
Beta-globin deletions (beta-FL, -619bp, Asian-Indian inversion)
Example of analysis: