Hindiii restriction endonuclease

HHV-6A and HHV-6B were differentiated according to the methodology described by Lyall and Cubie [65 (link)]. A two-step PCR reaction was performed again to differentiate between the two subtypes of the virus with primers targeting the HHV-6 large tegument protein (LTP) gene following HindIII restriction analysis. The primers used for the PCR reaction are summarized in Table 3. The obtained nPCR amplicons were digested with HindIII restriction endonuclease (Thermo Scientific, Waltham, MA, USA) according to the manufacture’s protocol. HindIII cleaves the HHV-6B positive sample into two fragments of 66 bp and 97 bp (base pair), but does not cleave HHV-6A at all. All PCR results were visualized using 1.7% agarose electrophoreses gel.

Obtained nPCR amplification products were digested with HindIII restriction endonuclease (Thermo Scientific, Waltham, MA, USA) which cleaves HHV-6B 163 bp amplification product into 66 bp and 97 bp fragments, whereas does not cleave HHV-6A [71 (link)].

Total genomic DNA (c.30 μg) was digested completely with the HindIII restriction endonuclease (Thermo), which recognizes a unique site within the T‐DNA region (Figure S1). Digested DNA was separated on 0.8% agarose gel and transferred to Hybond‐N⁺ nylon membrane (Amersham). A PCR‐generated bar gene fragment (primer 7 in Table S1) labelled with digoxigenin (DIG)‐High Prime (Roche) was used as a probe (Figure S1). Prehybridization, hybridization, membrane washing, and signal detection were carried out using DIG‐High Prime DNA Labeling and Detection Starter Kit II (Roche), according to the manufacturer's protocols.