Direct versus indirect detection of covalently closed circular (ccc)DNA. Direct versus indirect detection of covalently closed circular (ccc)DNA. (A) Southern.

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Direct versus indirect detection of covalently closed circular (ccc)DNA. Direct versus indirect detection of covalently closed circular (ccc)DNA. (A) Southern blotting. Vectors for wild-type (+ENV) and envelope-deficient (-ENV) duck HBV (DHBV) were transfected into the avian hepatoma cell line LMH. Viral DNAs from cytoplasmic nucleocapsids (cytopl) and in nuclear DNA were detected using a 32P-labeled DHBV DNA probe; nuclear DNAs were treated with Dpn I to digest bacterially derived transfected plasmid, and with Plasmid-Safe nuclease that degrades linear but not circular DNA; relaxed circular (RC)-DNA with a nearly completed (+)-strand is not degraded. Note (i) the characteristically distinct mobility of cccDNA from all other viral DNA forms and (ii) the drastically enhanced cccDNA levels generated from the envelope-deficient DHBV. (B) ‘cccDNA-specific PCR’. Primers (purple and green arrows) are chosen to span the region containing the gap in the (+)-strand and the nick in the (-)-strand of RC-DNA. cccDNA provides a continuous template for exponential amplification. On RC-DNA, linear extension of the individual primers generates shorter but overlapping products that can anneal to each other and subsequently form an identical amplicon as that from cccDNA. Michael Nassal Gut 2015;64:1972-1984 Copyright © BMJ Publishing Group Ltd & British Society of Gastroenterology. All rights reserved.