AEGiS-11IAC: Sequencing HIV isolates using the GeneChip HIV PRT assay.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Sequencing HIV isolates using the GeneChip HIV PRT assay.

Int Conf AIDS 1996 Jul 7-12; 11:8 (abstract no. Mo.A.164)
Miyada CG, Liang V, Tran HM, Mittman M, Morris M, Kaplan P; Affymetrix, Inc., Santa Clara, CA, USA. Fax: 408-481-0422. E-mail: garry_miyada@affymetrix.com.

OBJECTIVE: To develop a fast method to accurately sequence HIV isolates using a novel method of hybridization to high-density arrays of oligonucleotide probes.

METHODS: The GeneChip HIV PRT Assay includes a high-density oligonucleotide array with probes complementary to the protease gene and 242 amino terminal residues of the reverse of the reverse transcriptase gene. A sequence of 1041 bases is determined by hybridizing fluorescein-labeled RNA to the array. The array is manufactured by light-directed synthesis methods, contains over 11,000 distinct oligonucleotide probes, and measures 1.28 x 1.28 cm. Viral samples are amplified and cloned into plasmid vectors. The recombinant HIV clones are sequenced using both the GeneChip assay and automated dideoxy DNA sequencing.

RESULTS: Results from the GeneChip HIV PRT Assay and automated dideoxy DNA sequencing are compiled and compared for 12 clones. The average percentage of concordant calls per sample is 98.85% (range: 97.69-99.81%) and the average percentage of discordant calls per sample is 1.15% (range: 0.19-2.31%). Reverse transcriptase drug-resistance conferring mutations at codon 215 and neighboring codons are accurately identified in all cases (8 samples) using either technology.

CONCLUSION: The current configuration of the GeneChip assay sequences with an accuracy comparable to that obtained from automated dideoxy DNA sequencing. Starting with a single amplified sample, sequence data (1041 base pairs) is obtained within 3 hours. If steps are batched prior to hybridization, the assay has a throughput of 12 samples per day. For high-throughput sequencing applications, the GeneChip HIV PRT Assay offers high accuracy coupled with unprecedented speed.

Keywords: AEGIS, HIV, Hypoxanthine Phosphoribosyltransferase, HIV-1 Reverse Transcriptase, HIV-1, Reverse Transcriptase Inhibitors, HIV Protease, Anti-HIV Agents, HIV Infections, HIV Protease Inhibitors, RNA-Directed DNA Polymerase, Codon, Oligonucleotide Probes, Mutation, genetics, ICA11

960707
MoA164