Purification of the DNA polymerase of avian myeloblastosis virus

Adaptation of the Fluorogenic 5′-Nuclease Chemistry to a PCR-Based Reverse Transcriptase Assay

“…Given the specific activity of AMV RT and its molecular weight, this translates into a detection of between 8 and 80 molecules. Because there are between 20 and 100 RT molecules in a retrovirus particle (1,(4)(5)(6)11,16), the TM-PERT assay has the ability to detect a single virion in the 10 µ L of sample assayed. Thus, without concentrating the sample, this assay can detect retroviruses at a concentration of 100 virions per mL.…”

“…The cationic surfactant Catrimox-14 ™ (Iowa Biotechnology, Oakdale, IA, USA) is becoming a method of choice for extracting RNA from mammalian cells, tissues and biopsy samples (2)(3)(4)(5)(6)(7)(8). It rapidly lyses cells and complexes nucleic acids in a form that is not degradable by RNases.…”

“…The RNA can be recovered by extraction with either lithium chloride or hot buffered formamide, followed by ethanol-precipitation (3,4). In some circumstances, acidic guanidinium isothiocyanate (GITC) is used instead, followed by phenol-extraction and precipitation with isopropanol (2,(4)(5)(6)(7)(8). Catrimox has the added benefit that it effectively removes compounds that would otherwise inhibit polymerase chain reactions (PCRs) (9).…”

DNA polymerase activity in plasma from leukaemic guinea-pigs

“…Virus from plasma was purified by 3 cycles of low and high speed centrifugation, essentially as described by Bonar et al (1967). As a final step, the virus suspension was centrifuged at 95,000 q for 70 min through 20% glycerol in 041 M Tris-HCl pH 7 4, 0 05 M NaCl, 0.01 M EDTA (TNE pH 7.4) on to a pad of glycerol as described by Kacian et al (1971). The material on the glycerol pad was then pelleted at 95,000 g for 35 min, suspended in 0 05 M Tris-HCl pH 8-0, 006 M NaCl (RT buffer) or TNE pH 7-4 (as appropriate) and stored at -20TC.…”

Hybridization characteristics of enzymatically synthesised DNA complementary to mouse immunoglobulin messenger RNA

“…Avian myeloblastosis virus was obtained from the Microbiological Research Establishment, Porton Down, Wilts., and the RNA-dependent DNA polymerase was prepared by the method of Kacian et al [12]. 32 P-labelled eDNA was prepared using oe-[32 P]deoxyguanosine triphosphate (New England Nuclear, Boston, Mass., USA: specific activity 100 Ci/mmole; 10 ~tCi per incubation) in 50/ll volume containing 0.5/lg LE mRNA, 10/ll reverse transcriptase enzyme, 1 ~g oligothymidylic acid (p T~o, P.L.…”