AEGiS-11IAC: HIV-1 GeneChip and dideoxynucleotide sequence analysis of HIV-1 genomes present in plasma samples from patients of ACTG 143 study.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

HIV-1 GeneChip and dideoxynucleotide sequence analysis of HIV-1 genomes present in plasma samples from patients of ACTG 143 study.

Int Conf AIDS 1996 Jul 7-12; 11:221 (abstract no. Tu.A.265)
Mamtora G, Winters M, Drenkow J, Shafer R, Shen N, Tran H, Merigan T, Gingeras T; Affymetrix, Santa Clara, CA, USA. Fax: 408-481-0422.

OBJECTIVE: To correlate the nucleotide sequence of 22 HIV-1 genomes from ACTG 143 plasma samples obtained by high density oligonucleotide arrays (HIV-1 GeneChip assay) with conventional dideoxynucleotide sequencing results.

METHODS: A total of 0.2 ml of plasma from each patient was extracted for total nucleic acid using Phenol/Chloroform methodology. A 1.1 kb HIV-1-specific amplicon was produced by RT-PCR from each sample. A 1041 nucleotide region of this amplicon including all of the HIV-1 protease (PR) and 242 amino acids (aa) of the reverse transcriptase (RT) genes was sequenced by a collection of 8 to 11 dye terminator dideoxynucleotide sequencing reactions (using an 373 ABI gel reader) and by the HIV-1 GeneChip assay. The HIV-1 GeneChip contains greater than 11,000 different oligonucleotide probes synthesized on a 1.28 x1.28 cm glass substrate. The assay involves hybridizing fluorescein labeled HIV targets to the GeneChip arrays.

RESULTS: The HIV-1 GeneChip and the dideoxynucleotide sequencing methodologies each analyzed greater than 38,000 nucleotides of HIV-1 sequence from 44 plasma samples. The average concordance was 98.2% with the range of concordant results from 96.6% to 99.9%. Discordant results were categorized as unambiguous (62.4%) or ambiguous (37.6%). Many ambiguous discordances involve overlapping possibilities (e.g., dideoxynucleotide method calls a base C, T, or G and the GeneChip calls the same base a pyrimidine). Amplicons from several plasma samples were cloned and individual colonies (3-10) were analyzed by dideoxynucleotide sequencing. Clones derived from a single amplicon varied on average in 10 bases (range 4-20 bases) from the plasma sample derived sequence. Discordant calls at several nucleotide positions are explained by the presence of mixtures of genotypes represented at the interrogated position. A mutation at codon L74V which confers ddI resistance was detected in 9 of 21 post treatment samples by both methodologies. Interestingly, AZT conferring mutations at codons 41 (M to L) and 215 (T to Y) were also detected in 3 of the post treatment samples.

CONCLUSIONS: Both the HIV-1 GeneChip and dideoxynucleotide sequencing methodologies provide comparable genotyping results of HIV-1 genomes from plasma samples containing heterogeneous mixtures of virus. Resistance conferring mutations at codons L74V, M41L, and T215Y were concordantly identified by the 2 methods.

Keywords: AEGIS, HIV-1, HIV Protease, HIV-1 Reverse Transcriptase, Reverse Transcriptase Inhibitors, Sequence Analysis, Zidovudine, Anti-HIV Agents, HIV Protease Inhibitors, Genome, Codon, Didanosine, Mutation, RNA-Directed DNA Polymerase, Genotype, Oligonucleotide Probes, Human, genetics, ICA11

960707
TuA265