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Simple Preparation of Parapoxvirus Genome DNA for Endonuclease Analysis
Author(s) -
Inoshima Yasuo,
Morooka Akira,
Murakami Kenji,
Sentsui Hiroshi
Publication year - 2000
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2000.tb01247.x
Subject(s) - restriction enzyme , lysis , dna , alkaline lysis , dna extraction , biology , endonuclease , vaccinia , phenol extraction , virology , virus , ultracentrifuge , poxviridae , chromatography , sodium dodecyl sulfate , microbiology and biotechnology , chemistry , biochemistry , polymerase chain reaction , recombinant dna , plasmid , gene , rna , dna vaccination
We compared five methods for improved extraction of very‐large parapoxvirus DNA from infected cells: (i) alkaline‐lysis procedure followed by phenol extraction; (ii) modified Hirt procedure, which was a neutral lysis procedure followed by phenol extraction; (iii) Hirt procedure; (iv) method used for extraction of vaccinia virus DNA; and (v) standard procedure using virus purification with an ultracentrifuge and protease‐sodium dodecyl sulfate‐phenol treatment. The alkaline‐lysis procedure was more rapid, inexpensive and simpler than the other methods. Moreover, with this method it is not necessary to prepare any special facilities, reagents and kits. Although the extracted DNA was still crude, we could reproducibly prepare viral DNA from 2 × 10 6 infected cells in less than 2 hr and it could be readily digested by restriction endonuclease. This method will aid rapid genetic classification of parapoxvirus.