Rapid purification of hepatitis B virus DNA from serum.
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TLDR
It is shown, 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.Abstract:Ā
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.(ABSTRACT TRUNCATED AT 250 WORDS)read more
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References
More filters
Journal ArticleDOI
Detection of specific sequences among DNA fragments separated by gel electrophoresis.
TL;DR: This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters that can be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography.
Journal ArticleDOI
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase
Randall Keichi Saiki,David H. Gelfand,Susanne Stoffel,Stephen J. Scharf,Russell Higuchi,Glenn Thomas Horn,Kary B. Mullis,Henry A. Erlich +7 more
TL;DR: A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction, which significantly improves the specificity, yield, sensitivity, and length of products that can be amplified.
Journal ArticleDOI
Rapid and simple method for purification of nucleic acids.
R. Boom,C. J. A. Sol,M. M. M. Salimans,C. L. Jansen,P.M.E. Wertheim-van Dillen,J. van der Noordaa +5 more
TL;DR: A simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine, based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent.
Journal ArticleDOI
Avoiding false positives with PCR
S Kwok,Russell Higuchi +1 more
TL;DR: The exquisite sensitivity of the polymerase chain reaction means DNA contamination can ruin an entire experiment and adherence to a strict set of protocols can avoid disaster.
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