By Robert K. Hamatake, Johnson Y. N. Lau
A broad-ranging number of center concepts for the research of HBV and HDV infections and for the improvement of treatments to regard them. during this first of 2 volumes Detection, Genotypes, and Characterization, the authors concentrate on conveniently reproducible molecular equipment for the identity and quantification of viral markers, the detection and impression of viral variations, and the learn of the viral existence cycle. each one absolutely proven protocol is defined in step by step element via a longtime specialist within the box and contains a historical past advent outlining the main underlying the procedure, gear and reagent lists, and pointers on troubleshooting and heading off identified pitfalls. An accompanying moment quantity, Immunology, version structures, and scientific reviews, comprises straight forward protocols for the examine of host immune responses to an infection, in vitro and in vivo versions of an infection, and the advance of antivirals. For either volumes: A broad-ranging selection of center ideas for the examine of HBV and HDV infections and for the improvement of remedies to regard them. the 1st quantity Detection, Genotypes, and Characterization, the authors specialise in effortlessly reproducible molecular tools for the id and quantification of viral markers, the detection and impression of viral variations, and the learn of the viral existence cycle. the second one quantity, Immunology, version platforms, and scientific reports, comprises trouble-free protocols for the research of host immune responses to an infection, in vitro and in vivo versions of an infection, and the improvement of antivirals. each one absolutely confirmed protocol is defined in step by step element via a longtime professional within the box and incorporates a history creation outlining the main underlying the strategy, apparatus and reagent lists, and tips about troubleshooting and averting recognized pitfalls. The set deals either new and skilled investigators an encyclopedic number of strong instruments for learning HBV and HDV infections, a vital source for locating new cures to regard chronically contaminated sufferers.
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Extra resources for Hepatitis B and D Protocols: Volume 1: Detection, Genotypes, and Characterization
DNase I digestion protocol: a. 0 L serum nucleic acids. b. 4], 20 mM MgCl2). c. 0 L DNase I (1 U/L). d. Incubate at 25°C for 15 min. e. Stop reaction by adding 2 L ethylenediaminetetraacetic acid (EDTA) (25 mM). f. Inactivate enzyme at 65°C for 10 min. 2. In the one step RT–PCR, reverse transcription as well as PCR are performed without changing reagents between cDNA synthesis and ampliﬁcation. 5 L (100 ng/L) each of the primers 1445+ and 1574− is added, with a ﬁnal volume of 50 L. 3. Incubate tubes in a thermocycler at 50°C for 20 min (reverse transcription).
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 1434 + 1445 + 1454 + 1464 + 1485 − 1561 + 1574 − 1590 − 1668 − 1678 + 1683 a 1752 + 1806 a 1808 a 1824 − TCTCATCTGCCGGACCGTGT GGACCGTGTGCACTTCGCTT GCACTTCGCTTCACCTCTGC TCACCTCTGCACGTCGCATG TCCATGCGACGTGCAGAGGTGAAGC GACCGACCTTGAGGCATACTTCAAAGACTG CCTCAAGGTCGGTCGTTGAC CAGTCTTTGAAGTATGCCTCAAGGTCGGTC AATTTATGCCTACAGCCTCC ACCAGCACCATGCAACTTTT (T)15 GCTGG GTGCCTTGGGTGGCTTTAGGGCATGGACAT (T)15 AGCTC (T)15 GAAGC AGAGAGTAACTCCACAGAAG a Oligonucleotides in relation to f and tr RNA b Designations indicate the map positions of individual 5' ends (XhoI cordinates; 1) and polarities.
J. Virol. 71, 9392–9399. 50 Bowden et al. 9. Civitico, G. A. (1994) The half-life of duck hepatitis B virus supercoiled DNA in congenitally infected primary duck hepatocyte cultures. Virology 203, 81–89. 10. Locarnini, S. , Civitico, G. , and Newbold, J. E. (1996) Hepatitis B: new approaches for antiviral chemotherapy. Antiviral Chemistry Chemotherapy 7, 1–12. 11. Guidotti, L. , and Chisari, F. V. (1999) Viral clearance without destruction of infected cells during acute HBV infection. Science 284, 825–829.