GENETIC TESTING Volume 4, Number 1, 2000 Mary Ann Liebert, Inc. Arrayed Primer Extension: Solid-Phase Four-Color DNA Resequencing and Mutation Detection Technology ANTS KURG, 1 NEEME TÕNISSON, 1,4 IOANNIS GEORGIOU, 2 JOHN SHUMAKER, 3 JEFF TOLLETT, 3 and ANDRES METSPALU 1,4 ABSTRACT The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imag- ing, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 59 end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common b -thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identi- fied. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms. 1 INTRODUCTION G ENETICS AND MOLECULAR MEDICINE have an expanding need for rapid genotyping, mutation analysis, and DNA rese- quencing technologies that have a clear potential for miniatur- ization, parallelization, and automation and enable high throughput and ability to identify changes precisely in the pa- tient DNA. Conventional methods for mutation detection, such as single strand conformation polymorphism (SSCP), denatur- ing gradient gel electrophoresis (DGGE), chemical cleavage, or direct sequencing are time and labor intensive (Cotton et al., 1998) with little parallelization. A promising solution for this technological need is the use of oligonucleotide arrays using nucleic acid hybridization (Chee et al., 1996; Cronin et al., 1996; Hacia et al., 1996) or hybridization coupled with an en- zyme-mediated reaction, either by primer extension (Shumaker et al., 1996; Head et al., 1997; Pastinen et al., 1997) or liga- tion (Landegren et al., 1998). The most developed approach to- day, hybridization of labeled target to high-density oligonu- cleotide microarrays (e.g., Affymetrix GeneChip™ arrays), is a revolutionary method for DNA sequence analysis. Feasibil- ity studies of this approach are promising and the first results are impressive. However, the high complexity of the assays due to the large number of oligonucleotide probes per target se- quence base, sensitivity to hybridization conditions, compli- cated data analysis, and high cost are driving several research groups to look for alternative technologies. Here we present Arrayed Primer EXtension (APEX) tech- nology as an alternative to array-based DNA sequence analy- sis by hybridization. We describe an integrated system with chip and template preparation, multiplex primer extension on the ar- ray, fluorescence imaging, and data analysis. The method is based upon a two-dimensional (2D) array of oligonucleotides, immobilized via the 59 terminal amino group onto an epoxy- silanized glass support. This method can be viewed as dye-ter- minator sequencing of DNA, but instead of using one primer and analyzing hundreds of extension products in polyacryl- amide gel electrophoresis (PAGE), we can use hundreds to thousands of primers that are spatially separated and extend each by only one dye-labeled nucleotide. The single-tube sam- ple preparation protocol consists of PCR amplification followed by a DNA fragmentation reaction. After hybridization of the 1 Institute of Molecular and Cell Biology, Tartu Childrens Hospital, University of Tartu, Estonian Biocentre, Tartu 51010, Estonia. 2 Medical School, University of Ioannina, Ioannina 45500, Greece. 3 Baylor College of Medicine, Houston, TX, 77030. 4 Asper Ltd., Tartu 51014, Estonia.