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 dete ction of viral genomes with frequent mutations like HIV and hepatitis C virus. A system of 'stair' primers is suggested which allows amplifi cation 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 wh ich the 5' end remains constant (single-sized fragment) and the 3' end is displaced base by base. By PCR, 'stair' primers (HIV set) were com pared to single-sequence primers of 20 and 25 nucleotides chosen in th e same hypervariable: region of the HIV gp120 (on both sides of V3 reg ion), as well as to classical primers chosen in the conserved pol (pol V2) and gag (SK38-39) regions of the genome. Of 17 HIV isolates obtain ed by co-culture of lymphocytes from HIV-seropositive patients, 17/17 (100%) were amplified using stair primers. 14/17 (82%) with 25-nucleot ide 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 comparativel y 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 prime rs 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 detectio n of variable genomic regions of the HIV genome and amplification of s uch regions directly in patient leukocytes. In addition, the contribut ion of this system to microbiology and human genetics in general may b e important.