Quantification of HIV-1 proviral DNA and analysis of genomic diversity by polymerase chain reaction and temperature gradient gel electrophoresis.
A competitive polymerase chain reaction/temperature gradient gel electrophoresis (PCR/TGGE) protocol was developed for exact quantification of HIV-1 proviral DNA copy numbers in clinical samples. An internal standard (ST) that differs from wildtype-sequences only by a single base exchange was used as a competitor in PCR. Quantification of HIV-1 target sequences was achieved by coamplification of defined copy numbers of ST with wild type target sequences, hybridization of PCR products to a strand-specifically labelled probe, separation of ST and wildtype sequences by TGGE, and determination of the ratio of wildtype and standard sequences by densitometric scanning. Effects of sample preparation, DNA extraction and white blood cell counts were minimized by the additional quantification of beta-globin sequences. With this technique, it was possible to determine precisely the number of HIV target sequences as compared to the number of beta-globin gene copies with a detection limit of two HIV-1 proviral copies. Forty-four peripheral blood mononuclear cell (PBMC) extracts from 39 HIV-1 infected patients were analyzed by PCR/TGGE. HIV-1 proviral DNA levels ranged between 2 and 24190 HIV-copies/10(6) beta-globin copies. In general, patients in the advanced stages of disease and/or with low CD4 counts had much higher proviral DNA levels than patients in early stages or with high CD4 counts. In patients from whom consecutive samples were obtained, progression of disease correlated with a greater than tenfold rise of HIV-copies/10(6) beta-globin copies. Compared to other recently published protocols for proviral DNA quantification, this experimental approach allows in addition direct demonstration of mutations within the amplified region. The competitive PCR/TGGE protocol described in this study is suitable for monitoring fluctuations of proviral DNA levels and to identify the genomic diversity of HIV target sequences simultaneously in one assay.
Wieland
U
U
Institut fuer Virologie, Universitaet zu Koeln, Germany.
Suhr
H
H
Salzberger
B
B
Eggers
H J
HJ
Braun
R W
RW
Kühn
J E
JE
D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D014644:Genetic Variation; D016679:Genome, Viral; D005914:Globins / Q000235:genetics; D015658:HIV Infections / Q000097:blood / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics; D015203:Reproducibility of Results; D013696:Temperature
D004279:DNA, Viral; D005914:Globins