Amplification of the complete polyomavirus JC genome from brain, cerebrospinal fluid and urine using pre-PCR restriction enzyme digestion.
A method is described for amplification of the complete genome of the human polyomavirus JC (JCV) from progressive multifocal leukuencephalopathy (PML) brain, cerebrospinal fluid (CSF) of PML patients, and from the urine of controls without neurological disease. Efficient amplification with the 'long PCR' method is achieved with primers overlapping the single BamHI or EcoRI site following digestion of the DNA sample with the restriction enzyme BamHI or EcoRI, respectively. Cutting of the supercoiled JCV genome allows full separation of the strands during the denaturation step, and permits primer annealing to compete successfully with reassociation of the genomic DNA. With this method a single PCR amplification allows restriction fragment length polymorphism (RFLP) analysis and direct cycle sequencing of any part of the viral genome. RFLP analysis of JCV amplified from CSF has identified a new mutant sequence. Sequencing of clinical samples is useful for typing of JC virus isolates as Type 1 or Type 2 and for characterization of the JCV regulatory region as archetypal or rearranged.
Agostini
H T
HT
Laboratory of Experimental Neuropathology, NINDS, National Institutes of Health, Bethesda, MD 20892, USA.
Stoner
G L
GL
D001483:Base Sequence; D001921:Brain / Q000821:virology*; D002555:Cerebrospinal Fluid / Q000821:virology*; D017931:DNA Primers; D004278:DNA, Superhelical / Q000032:analysis / Q000302:isolation & purification; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D016679:Genome, Viral; D006801:Humans; D007577:JC Virus / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D014556:Urine / Q000821:virology*
D017931:DNA Primers; D004278:DNA, Superhelical; D004279:DNA, Viral; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI