By Feng Wang-Johanning, Gary L. Johanning (auth.), Paul M. Lieberman (eds.)
A compendium of quite simply reproducible and novel tips on how to manage DNA viruses and represent their diverse organic homes. The authors emphasize concepts for viral detection and genetics, but additionally contain tools for constitution selection, gene expression, replication, pathogenesis, complicated mobile types, recombinant genetics, and computational/systems techniques. Wide-ranging and hugely functional, DNA Viruses: equipment and Protocols will stimulate new instructions in virology study with its novel innovations for engineering viral vectors in gene remedy, and its complicated techniques for detecting viruses in human disorder.
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Additional resources for DNA Viruses: Methods and Protocols
A yet undeﬁned latency type with expression of EBER-1/2, EBNA-1, LMP-2a,b, BARTs, and BARF-1 is found in EBV-positive gastric carcinoma (5). BARF-1 mRNA expression is also detected in NPC and appears to be a carcinoma-speciﬁc marker not expressed in EBV-positive lymphomas (6,7). However, because BARF-1 mRNA is nonspliced, speciﬁc precautions are needed for its detection, as described in refs. 5–7. This chapter describes a sensitive reverse transcription polymerase chain reaction (RT-PCR) assay for determination of EBV latent RNA expression proﬁles in tumor biopsies or smears, isolated or cultured cells, and unfractionated whole blood.
Measure activity of wash buffer after the last washing step. If this is still active, additional washing steps may be performed until no activity is detected. 34 Stevens, Brink, and Middeldorp Fig. 1. Autoradiograph showing relative sensitivity of multiprimed EBV RT-PCR analysis. A dilution series of 1–100,000 EBV-positive JY cells was made in a whole blood DNA background, originating from an EBV-negative donor. JY is a lymphoblastoid cell line that expresses EBNA-1 mainly from the C/W promotor and shows limited ZEBRA expression in <1% of the cells.
G. BARTs (primers A3 and A4). h. ZEBRA (primers Z1 and Z2). i. The cellular housekeeping gene U1A snRNP (primers U1A1 and U1A2). 2 µL AmpliTaq DNA polymerase. Multiprimed EBV RT-PCR 33 4. Cycle the samples in a PCR device using the following PCR program: 4 min at 95°C; 40 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C; 7 min at 72°C, and ﬁnally a hold at 4°C (see Note 15). 4. 1. Electrophoretic Separation of PCR Products and Blotting to Nylon 1. 5% agarose gel electrophoresis in TBE buffer for 1–2 h at approx 100 mA.