Haplotypes of the human beta-like globin gene cluster: analyzing the 3' Hpa I polymorphic site by long distance PCR

Haplotypes of the beta-like globin gene cluster in individuals with sickle cell disease appear to be important as prognostic factors for the severity of the disease. The Hpa I polymorphic site 3' of the beta-globin gene is very often involved in haplotyping. However, this restriction site cannot be ascertained by conventional PCR, as it is in the middle of a long repetitive sequence. Long distance PCR was employed to amplify this Hpa I-containing region. By positioning either one or both primers outside the repetitive sequence, specific amplification was achieved. Moreover, sufficient amplification can be obtained by using a white cell lysate instead of purified DNA, thereby significantly reducing the amount of blood needed, and the total procedure time.