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Journal of Clinical Microbiology, July 2003, p. 3306-3311, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3306-3311.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Fluorescent Dye Terminator Sequencing Methods for Quantitative Determination of Replication Fitness of Human Immunodeficiency Virus Type 1 Containing the Codon 74 and 184 Mutations in Reverse Transcriptase

Viktoria Nurpeisov, Selwyn J. Hurwitz, and Prem L. Sharma^*

Department of Pediatrics and Laboratory of Biochemical Pharmacology, Emory University School of Medicine and Veterans Affairs Medical Center, Decatur, Georgia 30033

Received 9 December 2002/ Returned for modification 18 March 2003/ Accepted 11 April 2003

The fluorescent dye-labeled dideoxynucleotide automated DNA sequencing system has been routinely used for monitoring the development of resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) and protease genes during therapy. This system has provided information regarding the presence of mixtures of nucleotides in the clinical samples but has not previously been validated for the quantitative determination between peak heights and relative DNA concentration. We evaluated this system by using various ratios of wild-type and mutated DNA fragments and by performing sequencing reactions at actual melting temperatures of specific primers. Several different ratios of purified DNA fragments containing mixtures of L74/V74 and M184/V184 were sequenced, and peak heights were measured. Regression analysis between ratios of peak heights and DNA concentration demonstrated a statistically significant linear correlation, suggesting that the quantification of two different species of DNA in a mixture could be achieved with the fluorescent dye-labeled dideoxynucleotide system. These strategies have broader implications for the quantification of replication fitness of viruses, particularly those containing RT mutations at codons 74 and 184.

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* Corresponding author. Mailing address: Department of Pediatrics and Laboratory of Biochemical Pharmacology, Emory University and Veterans Affairs Medical Center, Medical Research 151H, 1670 Clairmont Rd., Decatur, GA 30033. Phone: (404) 321-6111, x7495. Fax: (404) 728-7726. E-mail: plsharm{at}emory.edu.

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Journal of Clinical Microbiology, July 2003, p. 3306-3311, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3306-3311.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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