3rd International Workshop on HIV Drug Resistance 2-5 August 1994, Kauai, Hawaii, USA

A NEW METHOD FOR RAPID SEQUENCE ANALYSIS: DETECTION OF HIV REVERSE TRANSCRIPTASE MUTATIONS

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:20 (abstract no. 19)

M.S. Chee, R. Yang, N. Shen, M. Kozal and T. Gingeras
Affymetrix, Santa Clara, CA, USA

We have developed DNA chips for the rapid sequence analysis of HIV reverse transcriptase. We have used the chips to identify and sequence mutations in drug resistant HIV strains, and are assessing the applicability of this approach to clinical monitoring of HIV drug resistance. The chips consist of ordered arrays of oligonucleotide probes synthesized on a glass substrate by repeated rounds of photodeprotection and coupling. Photolabile protecting groups are fIrst removed by exposure through a lithographic mask. Base monomers, themselves photoprotected, are then coupled to exposed sites using standard phosphoramidite chemistry. (Fodor SJ et al. Science. 1991 Feb 15;251(4995):767-73; Pease AC et al. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5022-6). Since the pattern of exposure directs the synthesis, any probe can be synthesized at any location. All probes length N, or subsets thereof, can be synthesized in only 4N or fewer steps. The HIV chip area is 1.28 × 1.28 cm, with probes in a 130 × 135 matrix. The size of each probe cell is approximately 98 × 95 microns. Nucleotides 2090 to 2946 (HIVBRU strain numbering) are represented on the chip with 4 sets of probes of different length: llmers, 13mers, 15mers and 17mers. In each set, each nucleotide position is represented by 4 probes. Each of the 4 probes contains a different base at the position to be sequenced: together they comprise a set of A, C, G and T probes which are otherwise identical. In principle, only one probe-target hybrid will be a perfect match. The other three will be single base mismatches. Fluorescence imaging of the hybridized chip allows quantification of hybridization signals.

An 831 bp fragment spanning the HIV reverse transcriptase coding sequence was amplifIed by PCR,using primers tagged with T3 and T7 promoter sequences. Labelled RNA was prepared by incorporation of fluorescent nucleotides, fragmented, and hybridized to chips. We analyzed viral clones and clinical samples and were able to identify drug resistance mutations and strain differences.

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1994-08-02
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