Genotypic resistance testing in HIV by arrayed primer extension.

@article{citeulike:2619062, abstract = {The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5'-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere.}, address = {Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany.}, author = {Schann\'{e}, Michaela and Bodem, Jochen and Gerhold-Ay, Aslihan and Jacob, Anette and Fellenberg, Kurt and Kr\"{a}usslich, Hans-Georg G. and Hoheisel, J\"{o}rg D. }, citeulike-article-id = {2619062}, doi = {http://dx.doi.org/10.1007/s00216-007-1775-0}, issn = {1618-2650}, journal = {Anal Bioanal Chem}, keywords = {genotyping, microarray, primer-design, probe-design, probe-selection, resequencing}, month = {January}, posted-at = {2008-04-01 10:33:24}, priority = {5}, title = {Genotypic resistance testing in HIV by arrayed primer extension.}, url = {http://dx.doi.org/10.1007/s00216-007-1775-0}, year = {2008} }

TY - JOUR ID - citeulike:2619062 L3 - citeulike-article-id:2619062 TI - Genotypic resistance testing in HIV by arrayed primer extension. JF - Anal Bioanal Chem AD - Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120, Heidelberg, Germany. SN - 1618-2650 N2 - The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5'-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere. KW - genotyping KW - microarray KW - primer-design KW - probe-design KW - probe-selection KW - resequencing AU - Schanné, Michaela AU - Bodem, Jochen AU - Gerhold-Ay, Aslihan AU - Jacob, Anette AU - Fellenberg, Kurt AU - Kräusslich, Hans-Georg AU - Hoheisel, Jörg PY - 2008/01/18/ UR - http://dx.doi.org/10.1007/s00216-007-1775-0 ER -