Polymerase chain reaction method for determining ratios of human immunodeficiency virus proviral DNA to cellular genomic DNA in brain tissues of HIV-infected patients.
A PCR method was developed to compare HIV-1 DNA loads in brain tissue samples. The method determines the ratio of the amplified product of an HIV DNA sequence to that of a host cellular DNA sequence using standard DNAs as reference. The standards include DNA from a line of human cells that harbor one HIV-1 provirus per cellular genome, and DNA from non-infected human cells. The standard DNAs were mixed in varying proportions and used to establish conditions of amplification under which the ratios of their PCR-amplified products corresponded with the ratios of the amounts of the DNAs themselves. The method was evaluated using known mixtures of the standard DNAs. Using the conditions thus obtained, ratios of HIV proviral DNA to cellular genomic DNA were obtained for tissue DNA samples taken from several different locations within the brain of two deceased HIV-infected patients. Results showed that HIV DNA was non-uniformly distributed within each brain (10-250 per 10(3) cellular genomes); the highest ratios were found in the hippocampus for each patient, independent of postmortem neuropathological findings. The criteria for quantitative PCR have general applicability to comparative studies of any proviral DNA loads in different tissue samples.
Fujimura
R K
RK
Molecular Biology Branch, Food and Drug Administration, Rockville, MD 20857, USA.
Bockstahler
L E
LE
D000163:Acquired Immunodeficiency Syndrome / Q000821:virology; D000199:Actins / Q000235:genetics; D001483:Base Sequence; D001921:Brain / Q000473:pathology / Q000821:virology*; D001927:Brain Diseases / Q000821:virology; D017931:DNA Primers; D004279:DNA, Viral / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D015683:Gene Products, gag / Q000235:genetics; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics
D000199:Actins; D017931:DNA Primers; D004279:DNA, Viral; D015683:Gene Products, gag