New types of primers (stair primers) for PCR amplification of the variable V3 region of the human immunodeficiency virus.
The aim of the study was to develop a reliable PCR method for the detection of viral genomes with frequent mutations like HIV and hepatitis C virus. A system of 'stair' primers is suggested which allows amplification of a genomic sequence despite the presence of mutations in the region of the primers. In this system, classical primers are replaced with primers composed of a mixture of equimolar oligonucleotides in which the 5' end remains constant (single sized fragment) and the 3' end is displaced base by base. By PCR, 'stair' primers (HIV set) were compared to single-sequence primers of 20 and 25 nucleotides chosen in the same hypervariable region of the HIV gp120 (on both sides of V3 region), as well as to classical primers chosen in the conserved pol (polV2) and gag (SK38-39) regions of the genome. Of 17 HIV isolates obtained by co-culture of lymphocytes from HIV-seropositive patients, 17/17 (100%) were amplified using stair primers, 14/17 (82%) with 25-nucleotide primers, and 12/17 (70%) with 20-nucleotide primers. Amplification occurred in 17/17 instances with polV2 primers and in 16/17 instances with SK38-39. In addition, 55 other isolates were tested comparatively using stair, polV2 and SK38-39 primers. All isolates were amplified using stair and SK38-39 primers and 54/55 isolates with polV2 primers. When applied to 22 extracts of patients' lymphocytes DNA, stair primers amplified all 22 extracts to the same degree as polV2 and SK38-39, whereas the 20 and 25 nucleotide primers chosen in the variable region were not as reliable. This new primer system allows reliable detection of variable genomic regions of the HIV genome and amplification of such regions directly in patient leukocytes. In addition, the contribution of this system to microbiology and human genetics in general may be important.
Colimon
R
R
Laboratoire de Bactériologie-Virologie, Faculté de Médecine de Rennes I, France. ronald.colimon@univ-rennesl.fr
Minjolle
S
S
André
P
P
de la Pintière
C T
CT
Ruffault
A
A
Michelet
C
C
Cartier
F
F
D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D004279:DNA, Viral; D015723:Gene Library; D006678:HIV / Q000235:genetics / Q000302:isolation & purification*; D015699:HIV Envelope Protein gp120 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D004279:DNA, Viral; D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments