RAPID PURIFICATION OF HEPATITIS-B VIRUS-DNA FROM SERUM

We describe two rapid, simple, and reliable procedures for routine purification of hepatitis B virus (HBV) DNA from serum. HBV DNA could be purified from 24 serum samples in 1.5 to 2 h and was recovered in the initial reaction vessel. Both procedures have in common that HBV DNA is complexed with silica particles in the chaotropic agent guanidinium thiocyanate (GuSCN) but differ in lysis conditions and in the conditions used to elute HBV DNA from the silica particles after purification of the silica-DNA complexes. In one procedure (protocol H), serum HBV lysis was mediated by sodium dodecyl sulfate-proteinase treatment and HBV DNA was subsequently complexed with silica particles in the presence of GuSCN. After washing and drying of the silica-DNA complexes, HBV DNA was eluted from the silica particles in a low-salt buffer. In the other procedure (protocol Y*), serum HBV was directly lysed in GuSCN and HBV DNA was simultaneously complexed with silica particles. After washing and drying of the complexes, HBV DNA was eluted by proteinase treatment in low-salt buffer. Omission of proteinase treatment prevented efficient elution, presumably because of copurification of the protein which is covalently bound to the HBV DNA genome. We show, by Southern blot analysis, that HBV DNA could be reproducibly purified from human serum with the same yields by either procedure (30 to 50% relative to a classic procedure) and apparently independent of serum composition. HBV DNA purified by either method was a good substrate in the polymerase chain reaction compared with DNA purified by the classic procedure. HBV DNA purified by protocol H could efficiently be repaired by Klenow DNA polymerase, which resulted in the generation of a discrete linear DNA molecule of 3.4 kb, regardless of the complexity of the hybridization pattern of unrepaired HBV DNA. The 3.4-kb DNA molecule could subsequently be cleaved by restriction enzymes into discrete fragments adding up to 3.4 kb. Fragment lengths were in accordance with those expected for a repaired linear HBV DNA molecule which was derived from the circular HBV DNA genome by opening up at a fixed point, presumably to cohesive end region. The ease and rapidity of the latter procedure may allow large-scale epidemiologic studies of the HBV genome by restriction enzyme analysis.