Genotypic Assays


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Genotypic Assays

Several approaches to genotyping are available, ranging from full-length sequencing of the target gene to point mutation assays, which focus only on a particular mutation of interest. The most commonly used genotypic assays rely on automated DNA sequencing. Using this technique, the nucleotide sequence of some or all of the gene of interest (e.g., protease [PR] or reverse transcriptase [RT]) is obtained, then translated into the predicted amino acid sequence in order to determine whether specific mutations are present or absent. Automated sequencing offers the most complete data on viral genotype, but generates more information than is needed for most clinical purposes. For example, HIV-1 RT has 550 amino acids, but mutations at only a small number of these positions are implicated in drug resistance. Therefore, interpretation of the genotype is needed in order to help distinguish which changes are merely polymorphisms and which might be significantly associated with drug resistance.

Most commercially available genotypic tests rely on automated sequencing technology. Viral RNA is extracted from a sample of plasma and reverse transcribed into complementary DNA in the laboratory. The PR- and RT-coding regions of the cDNA are then amplified by polymerase chain reaction (PCR), and the nucleotide sequence of the PCR product (or amplicon) is determined on an automated DNA sequencer. Some laboratories use specific kits developed by companies such as ABI/Perkin Elmer or Visible Genetics, Inc. to perform genotyping. Usually the kits provide standardized reagents needed for the RT-PCR and DNA sequencing steps. Other laboratories use so-called "home brew" assays using reagents and primers developed individually by each laboratory. A list of the mutations most often associated with resistance to currently available drugs is given in Table 1.

Other types of genotypic resistance assays such as the Line Probe Assay (LiPA; Innogenetics) (3) or the differential probe hybridization assay being developed by Bayer (4) are designed to provide more limited information by testing for the presence or absence of specific mutations at particular positions, or codons. These assays have the advantage of being faster and less complex than standard genotyping, and may be more sensitive at detecting minor species. However, because these tests do not generate a comprehensive sequence, information needed to interpret complex genotypes might be missing. Furthermore, the tests must be reconfigured to include important new mutations as they are defined.

4/15/01