Detection of Moloney murine sarcoma virus in tissues and cultured cells by the polymerase chain reaction.
The polymerase chain reaction was used for Moloney murine sarcoma virus (MoMuSV) detection in frozen and formalin-fixed, paraffin-embedded tissue sections and cultured cells isolated from MoMuSV-induced tumors. Rapid DNA extraction by proteinase K digestion, followed by CHROMA SPIN + TE-100 column purification proved to be satisfactory. Two pairs of overlapping primers, flanking 1026 base pair (bp) to 221 bp, allowed to choose among four different length of DNA-amplified segments. Although net amplification was obtained for frozen tissue and tumor cultured cells in all combinations of primers, the maximum specificity and sensitivity resulted with 602 bp fragment. This product was fully and adequately digestible using Apa I and Sau3A I restriction endonucleases. DNA extracted from paraffin-embedded sections yielded an amplification product only when the primer pair which delineated a 221-bp segment was used. This reproducible method could be useful for diagnostic and for pathogenetic investigations of MoMuSV infections.
Tasca
S I
SI
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station 77843.
Hoffman
R J
RJ
Stoica
G
G
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004277:DNA, Single-Stranded / Q000737:chemistry; D004279:DNA, Viral / Q000302:isolation & purification*; D015252:Deoxyribonucleases, Type II Site-Specific; D004195:Disease Models, Animal; D004587:Electrophoresis, Agar Gel; D005629:Frozen Sections; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008980:Moloney murine sarcoma virus / Q000235:genetics / Q000302:isolation & purification*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011533:Proviruses / Q000235:genetics; D012513:Sarcoma, Experimental / Q000382:microbiology / Q000473:pathology; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases; C038485:GGGCCC-specific type II deoxyribonucleases